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Gibberellin-like substances were found to be produced by fungi isolated from mycorrhizae of pine. Among the isolates studied gibberellin-like substances producers were most numerous among the Basidiomycetes forming ectotrophic mycorrhiza and among non-sporulating fungi forming no mycorrhiza. In most strains the gibberellin-like subtances were elaborated after 10--20 days of incubation. The Rf values of the gibberellin-like compounds were different in various strains, but in most of them substances showing gibberellin activity appeared on the chromatograms run with benzene, acetic acid (10:3 v/v) at Rf 0.2--0.6.

The previously reported inability of methionine to stimulate cephalosporin C production in a cysteine auxotroph is due to cysteine interference with methionine uptake. In such a cse, "illicit transport" of alanylmethionine can be used to demonstrate the efficacy of methionine in such mutants blocked in the path from methionine to cysteine. This result supports the hypothesis that the stimulatory activity of methionine is not due to its ability to donate sulfur to the caphalosporin C molecule.

A new metabolite was isolated from the culture filtrate of a deacetylcephalosporin C-producing mutant, derived from Cephalosporium acremonium ATCC 14553, by means of adsorption on activated carbon, column chromatography on DEAE-Sephadex A-25 and gel filtration through a Sephadex G-10 column. The compound was identified as D-5-amino-5-carboxyvaleramido-(5-formyl-4-carboxy-2H, 3H, 6H-tetrahydro-1, 3-thiazinyl) glycine by spectral analyses, elucidation of hydrolysis products of the compound, and comparison of characteristics of the compound with those of a synthetic authentic compound.

A mutant with enhanced potential to utilize sulfate for cephalosporin C production was isolated from a strain of Cephalosporium acremonium. The mutant displayed potency levels more than twofold that of the parent in the presence of sulfate but its productivity was severely inhibited by more than 0.5% of methionine which gave high cephalosporin C production with the parent. In a complex medium norleucine stimulated cephalosporin C production by the mutant in the presence of sulfate, whereas it showed no effect on the parent. In an incubation system with sulfur-starved cells of the mutant, L-methionine, but not the D-isomer, gave lower cephalosporin C production and a delayed production of penicillin N. However, it exhibited a stimulatory effect in the presence of valine or alpha-aminoadipic acid, the constituent amino acids of the antibiotic. Norleucine showed a similar effect to that of L-methionine in the presence of sulfate. On the basis of these results, characteristics of the mutant are discussed in connection with the effect of methionine.

A number of Cephalosporium acremonium mutants blocked in the synthesis of cephalosporin C were investigated for accumulation of other beta-lactam compounds. The non-cephalosporin C producers were isolated after exposing the superior cephalosporin C-producing strain M8650-4 to ultraviolet light (268 nm). One of the blocked mutants, MH63, accumulated deacetylcephalosporin C (0.4 mg/ml), deacetoxycephalosporin C (1.5 mg/ml), and penicillin N (2.7 mg/ml). In contrast, the parent of MH63 produced high levels of cephalosporin C as well as deacetylcephalosporin C (2.2 mg/ml) and penicillin N (1.0 mg/ml), but only traces of deacetoxycephalosporin C (about 0.1 mg/ml). Deacetoxycephalosporin C was isolated from the mutant strain and identified by ultraviolet light, nuclear magnetic resonance, bioactivity spectrum, and co-migration with authentic standard in three chromatography systems.

Conditions for the accurate measure of glutamic dehydrogenase (GDH) from Cephalosporium acremonium were determined. K(m) values for alpha-ketoglutarate and ammonium ion were 7 and 15 mM, respectively. The half-saturation for reduced nicotinamide adenine dinucleotide phosphate was 5 muM. Reduced nicotinamide adenine dinucleotide did not serve as a cofactor for the enzyme. The specific activity of GDH was measured in six mutants of C. acremonium which varied in their ability to synthesize cephalosporin C. The mutants represented two separately derived lines, A and B. The four mutants in line B were characterized by a derepression of the GDH upon entry into stationary phase. The two mutants in line A were characterized by repressed levels of GDH during the same period. Both lines exhibited high GDH activity early in their fermentations, but activity decreased during the period of active cell growth. Cytochrome c concentrations followed the same pattern as total soluble intracellular protein. Line A mutants were low in cephalosporin C productivity and line B encompassed low, intermediate, and high productivity mutants. The relative frequency of yield improvements in line A and B indicate that the altered regulation pattern for GDH in line B may have removed a nitrogen limitation for cephalosporin C synthesis.

Differentiation of swollen hyphal fragments to unicellular arthrospores accompanied the synthesis of cephalosporin C by a series of Cephalosporium acremonium mutants during propagation in a complex medium. The complex medium supported significantly higher synthesis than the defined medium used in previous studies of differentiation in C. acremonium. The mutants differed in their ability to form unicellular arthrospores and to synthesize cephalosporin C, but a one-to-one correspondence between the two properties was not observed. An inverse relation was observed between the growth rates of the mutants and their ability to synthesize cephalosporin C: each mutant produced more antibiotic but grew more slowly than its parent strain. Germination of the unicellular arthrospores occurred in complex medium but differed significantly from the germination of conidia in seed medium. The unicellular arthrospores were examined by electron microscopy and compared with swollen hyphal fragments and slender hyphal filaments. The unicellular arthrospores had a thicker cell wall, rougher cell surface, and had one or more small identations in their surface. The internal structure of the unicellular arthrospore resembled those of the swollen hyphal fragment and slender hyphal filament. Filaments had lower concentrations of lipid-containing vacuoles which were prevalent in both the swollen hyphal fragments and the unicellular arthrospores.

An early blocked sulfur amino acid auxotroph, Cephalosporium acremonium mutant 274-1 (which could be satisfied by methionine or cysteine), utilized organic sulfur compounds for cephalosporin C production in the following order of decreasing effectiveness; methionine > cystathionine > cysteine, despite the fact that cysteine is considered to be the immediate precursor of the antibiotic. When a genetic block was added to mutant 274-1 in the transsulfuration pathway from cysteine to methionine, the double mutant 11-8 (which grows on methonine but not cysteine) failed to produce cephalosporin C from cysteine even though enough methionine was added to support normal growth. Addition of the non-sulfur analogue, norleucine, resulted in antibiotic production from cysteine in the double mutant. These facts support the hypothesis that methionine stimulation of cephalosporin C production is due to a role of methionine other than that of sulfur donation.

1. Three intracellular peptides found in small amount in a Cephalosporium sp. were rapidly labelled when dl-[(14)C]valine was added to a shaken suspension of the organism. More (14)C was incorporated into peptide P3, delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine, than into peptide P2 (containing alpha-aminoadipic acid, cysteine, valine and glycine) or peptide P1 (containing beta-hydroxyvaline in place of the valine in peptide P2). 2. Peptides P3 and P2, but not peptide P1 were formed in a broken-cell system from the Cephalosporium sp. in the presence of delta-(l-alpha-aminoadipyl)-l-cysteine and dl-[(14)C]valine. No synthesis was observed in the presence of delta-(d-alpha-aminoadipyl)-l-cysteine or of dl-alpha-amino[(14)C]adipic acid and l-cysteinyl-l-valine or l-cysteinyl-d-valine. 3. The biosynthesis of these peptides was catalysed by the particulate fraction of the broken-cell system, whereas that of glutathione was catalysed by the supernatant fraction. 4. These results are discussed in relation to penicillin N and cephalosporin C biosynthesis.

In submerged cultures, Cephalosporium acremonium exists in four morphological forms: hyphae, arthrospores, conidia, and germlings. The phase of hyphal differentiation into arthrospores coincides with the maximum rate of beta-lactam antibiotic synthesis. Furthermore, arthrospores, separated by density-gradient centrifugation, possess 40% greater antibiotic-producing activity than any other morphological cell type. In a series of mutants, each with an increased potential to produce beta-lactam antibiotics, differentiation into arthrospores was proportional to the increased titer of these antibiotics. Thus, arthrospores exhibit enhanced synthesis of beta-lactam antibiotics and appear to be a determining factor in high-yielding mutants. Since a non-antibiotic-producing mutant readily differentiated into arthrospores, antibiotic synthesis and cellular differentiation are not obligately related.

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.

Aphidicolin is an antibiotic of novel structure produced by the mold Cephalosporium aphidicola. It is a potent inhibitor of cellular deoxyribonucleic acid synthesis, and it also strongly inhibits the growth of herpes simplex virus both in tissue culture and in the rabbit eye. Aphidicolin is active against iododeoxyuridine-resistant herpes virus, and does not itself readily induce the formation of drug-resistant strains of herpesvirus.

Eighty-eight strains of microorganisms were isolated from soils collected in northern and southern Chile, and 10 fungi which showed the highest solubilizing action upon the iron in granodiorite were then selected. These fungi were incubated with the following iron-containing minerals: augite, hornblende, biotite, magnetite, hematite, and the igneous rock granodiorite. The solubility of iron in these minerals depended on their nature, crystalline structure, the concentration of metabolic products, or all three. Complex formation could be the mechanism involved, as a strong cation-exchange resin was not able to extract Fe from culture solutions. This conclusion is also confirmed by the R(F) values obtained by thin-layer chromatography of iron-containing culture solutions.

Three aggregational forms of arylamidase are produced by Cephalosporium acremonium. The exocellular enzyme, with an approximate molecular weight of 60,000, was purified 300-fold by diethylaminoethyl cellulose chromatography, gel filtration, and gel electrophoresis. With l-leucyl-beta-naphthylamide as the substrate, the K(m) is 4.2 x 10(-4)m; the optimum pH, 7.7; and the temperature optimum, 35 C. The enzymatic hydrolysis of l-leucyl-beta-naphthylamide is inhibited by a number of cephalosporins, whereas a variety of penicillins show no effect. Alternatively, the enzyme specifically catalyzes the beta-lactam hydrolysis of a number of cephalosporins; a number of penicillins are resistant. The K(m) for cephalosporin C is 9.09 x 10(-4)m.

The 12,13-epoxytrichothecenes, a group of sesquiterpenoid fungal antibiotics, inhibit protein synthesis in eukaryotic cells but do not share a common mode of action. Trichodermin stabilizes polyribosomes, prevents their disaggregation by puromycin, and also prevents the release of nascent peptides from ribosomes by puromycin. Nivalenol, T-2 toxin, and verrucarin A cause rapid and almost quantitative breakdown of polyribosomes in H-HeLa cells, a process which is inhibited by anisomycin, cycloheximide, or trichodermin. Similar effects of trichodermin, nivalenol, and verrucarin A are also observed in yeast spheroplasts. We conclude that nivalenol, T-2 toxin, and verrucarin A are potent and highly selective inhibitors of polypeptide chain initiation in eukaryotes, whereas trichodermin inhibits chain elongation and (or) termination. We have compared the structural formulae of various trichothecenes and suggest that the presence of substituents on carbon-15 of the common trichothecene ring may be important in determining the precise modes of action of this group of compounds.

Methionine has an almost unique stimulatory effect on biosynthesis of cephalosporins (by Cephalosporium acremonium). No other sulfur-containing compound tested, except dl-methionine-dl-sulfoxide, replaced methionine. dl-Methionine stimulated the synthesis of cephalosporins when added after the growth phase. The utilization of inorganic sulfate was repressed by methionine. Experiments with l-methionine-S(35) showed that essentially all the sulfur in the cephalosporins was derived from methionine. Sulfur-labeled compounds found in the soluble pool from cells grown with methionine-S(35) were methionine, homocysteine, taurine, cystathionine, cysteic acid, glutathionine, and cysteine. dl-Serine-3-C(14) was incorporated into the antibiotics, and its utilization was stimulated by methionine. l-Cysteine had a sparing effect on the incorporation of methionine-S(35) and serine-C(14) into the antibiotics. The data are consistent with the hypothesis that a cystathionine-mediated pathway is operative in the transfer of sulfur between methionine and cysteine.

1. l-alpha-Amino[6-(14)C]adipic acid has been prepared from the dl-amino acid by oxidation of the l-isomer with l-amino acid oxidase to alpha-oxo[6-(14)C]adipic acid and by transamination of the latter with l-glutamic acid in an extract of a Cephalosporium sp. prepared by ultrasonic treatment of the mycelium. 2. The optical configuration of small amounts of (14)C-labelled alpha-aminoadipic acid from the mycelium of the Cephalosporium sp. has been determined by treatment with l-amino acid oxidase and measurement of the proportion of radioactivity subsequently retained on a column of a strong cation-exchange resin. 3. alpha-Aminoadipic acid which had been labelled in the mycelium from [1-(14)C]acetate appeared to contain more than 99% of the l-isomer. 4. l-alpha-Amino[(14)C]adipic acid (sodium salt) was taken up much more rapidly than the d-isomer, or alpha-oxo[6-(14)C]adipic acid, by suspensions of washed mycelium of the Cephalosporium sp. in water. The pool of intracellular alpha-aminoadipic acid was expandable. 5. Intracellular products found to be labelled with (14)C from l-alpha-amino[(14)C]adipic acid were delta-aminovaleric acid, saccharopine, lysine, protein, compounds which behaved like penicillin N, cephalosporin C and deacetylcephalosporin C respectively on paper chromatography and electrophoresis, and a peptide whose amino acid residues include alpha-aminoadipic acid, cysteine and valine. 6. l-alpha-Amino[(14)C]adipic acid acted as a precursor of the delta-(d-alpha-aminoadipoyl) side chains of extracellular penicillin N and cephalosporin C. 7. (14)C from d-alpha-amino[(14)C]adipic acid was incorporated into penicillin N and cephalosporin C, but the incorporation was accompanied by a relatively high dilution of specific radioactivity and some l-alpha-amino[(14)C]adipic acid was found in the intracellular pool. 8. These findings are discussed in relation to the origin of the d- configuration of the alpha-aminoadipoyl side chain of the antibiotics.