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Cell-free preparations of both Rhizoctonia solani, a sterol-synthesizing fungus, and Phytophthora cinnamomi, a non-sterol-synthesizing fungus, incubated in the presence of [2(-14)C]mevalonate and iodacetamide, converted the mevalonate into labelled mevalonate 5-phosphate, mevalonate 5-pyrophosphate and isopentenyl pyrophosphate. In the absence of iodoacetamide, but under anaerobic conditions, the same preparations converted the mevalonate into labelled geraniol, farnesol and squalene, the first two compounds presumably as their pyrophosphates. When cell-free preparations of both organisms were incubated aerobically in the presence of [1(-14)C]isopentenyl pyrophosphate, only labelled geraniol, farnesol and squalene were recovered from the P. cinnamomi reaction mixture, whereas labelled geraniol, farnesol, squalene, squalene epoxide, lanosterol and ergosterol were present in the R. solani reaction mixture. When these same preparations were incubated in the presence of 14C-labelled squalene, labelled squalene epoxide, lanosterol and ergosterol were recovered from the R. solani reaction mixture. In contrast, the P. cinnamomi preparation was unable to convert the squalene into products further along the sterol pathway; instead, a portion of the labelled squalene was converted into water-soluble products, indicating the possible existence of a squalene-degradation process in this organism. It appears that the block in the sterol biosynthetic pathway of P. cinnamomi occurs at the level of squalene epoxidation.

Phytophthora cinnamomi, a member of the Pythiacease, does not synthesize sterols. Small amounts of squalene, but no squalene epoxide or sterol, were isolated from the dried mycelium of this fungus after growth in sterol-free medium. The dried mycelium of Rhizoctonia solani, a sterol-synthesizing fungus grown under the same conditions, contained small amounts of squalene and squalene epoxide and large amounts of ergosterol. When the two organisms were grown in the presence of [14C]acetate, only labelled geraniol, farnesol and squalene were recovered from the P. cinnamomi mycelium, whereas labelled geraniol, farnesol, squalene, squalene epoxide and ergosterol were recovered from the R. solani mycelium. Similar results were obtained when the organisms were incubated in the presence of [2(-14)C]mevalonate; in this case, labelled lanosterol was also detected in the R. solani mycelium. Both organisms, when incubated in the presence of unlabelled squalene, squalene epoxide or lanosterol, incorporated these compounds into their mycelia; however, only the R. solani mycelium was able to convert these substrates into products further along the sterol pathway. It appears that squalene is the terminal compound in the sterol biosynthetic pathway of P. cinnamomi.

Peripheral hyphae were separated from the remaining thallus of Rhizoctonia solani in exponential and stationary phases of growth. The QO(2) in whole cells of peripheral hyphae from young fungal colonies was on the average 2.6 times and the protein content 1.6 times greater than in peripheral hyphae from old fungal colonies. The overall rate of amino acid uptake was less in old than in young fungal colonies. In a polyuridylic acid-polyphenylalanine incorporating system, the two kinds of peripheral hyphae required ribosomes, supernatant fraction, polyuridylic acid, soluble ribonucleic acid, adenosine triphosphate, and pyruvate kinase. The rate of polyphenylalanine synthesis in old fungal colonies was slower than in the young fungal colonies. The ribosomes and supernatant fraction of the young and old fungal colonies were interchangeable and active. The factor responsible for deficient protein synthesis in old fungal colonies appears to be in the soluble fraction of the mycelium.

A study was made of the ability of cell-free protein synthesis systems from vegetative cells of different age of the fungus Rhizoctonia solani to produce polyphenylalanine. Polyuridylic acid-directed phenylalanine incorporation into peptides decreased linearly with cell age. The 105,000 x g supernatant fluid and ribosomal fractions were equally responsible for the total loss of synthetic activity of the older cells. Initial rates of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase activity decreased with increasing cell age, which accounted for the defect of the supernatant fraction. An accelerated degradation of soluble phenylalanyl-RNA was associated with the ribosomes of the older cells. In vitro systems from cells of different age transferred phenylalanine from phenylalanyl-tRNA to polyphenylalanine at similar rates. Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 increased and 5 decreased in specific activity with increased age; 3 others did not change during aging and 2 were below acceptable detectable levels.

Dowler, William M. (University of Illinois, Urbana), Paul D. Shaw, and David Gottlieb. Terminal oxidation in cell-free extracts of fungi. J. Bacteriol. 86:9-17. 1963.-The terminal respiration in cell-free extracts of ten representative fungi is mediated by an electron transport system similar to that observed in animal tissue. Reduced nicotinamide adenine dinucleotide (NADH) and succinic cytochrome c reductases and NADH oxidase activity are contained in the extracts. An antimycin-sensitive site and cytochrome oxidase are present. Dehydrogenases including glucose-6-phosphate, triose phosphate, isocitric, glutamic, succinic, and malic dehydrogenase were found, but pyruvic and alpha-ketoglutaric dehydrogenases were absent. The soluble nature of the dehydrogenases indicates probable disruption of the mitochondria, since normally these enzymes are found within the mitochondria. Particles containing most of the terminal respiratory activity could be sedimented by centrifugation at 40,000 x g for 30 min. Oxygen was utilized by cell-free extracts with NADH, succinate, and isocitrate as substrates, but not with glucose.

Schlosser, Eckart (University of Illinois, Urbana), and David Gottlieb. Sterols and the sensitivity of Pythium species to filipin. J. Bacteriol. 91:1080-1084. 1966.-The growth of several Pythium species was not affected by filipin. No leakage of inorganic phosphate was observed after treatment with the antibiotic. No sterol could be detected in 1 g (dry weight) of mycelium. Thus, the insensitivity of these fungi to the antibiotic may be explained by the lack of sterols, the postulated reaction site for filipin in the cell membrane. Though not capable of synthesizing sterols, Pythium species can incorporate exogeneous sterols, which renders them sensitive to filipin; such treatment causes a lag in growth and leakage of inorganic phosphate. The leakage after filipin treatment is indirect evidence that the sterols have been incorporated into the cell membrane. Induced sensitivity to filipin was reversible; it was lost when the sterols were diluted out by one transfer through a medium free from sterols. The hypothesis that the primary site of interaction of filipin is the sterol located in the cell membrane was strengthened by these studies. The experiments further demonstrated a change in sensitivity of a fungus to a toxic agent due to nutritional conditions.

Gottlieb, David (University of Illinois, Urbana), and James L. Van Etten. Changes in fungi with age. I. Chemical composition of Rhizoctonia solani and Sclerotium bataticola. J. Bacteriol. 91:161-168. 1966.-The chemical composition of the mycelium of Rhizoctonia solani and Sclerotium bataticola was determined in cells of various ages. The percentage, per unit of dry weight, of soluble amino nitrogen, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), ergosterol, and protein decreased with age in both fungi. Total lipids and fatty acids increased with age in S. bataticola but remained constant in R. solani. Total carbohydrate increased with age in R. solani and decreased in S. bataticola. Fewer changes with age were observed when the results were calculated in ratio to DNA. There was no change in the ratios of protein, RNA, and soluble amino nitrogen to DNA with age in either fungus, but the ergosterol-DNA ratio decreased. The total lipid-DNA ratio and the total fatty acid-DNA ratio increased with age in both fungi, whereas the total carbohydrate-DNA ratio increased in R. solani but remained constant in S. bataticola. Both fungi contained myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acids. In addition, R. solani contained pentadecanoic acid, and S. bataticola had myristoleic, linolenic, and arachidic acids. No marked change in the fatty acid pattern of S. bataticola was observed with age, whereas in R. solani the percentage of linoleic acid per total fatty acids decreased slightly when oleic acid increased.

Gottlieb, David (University of Illinois, Urbana), and James L. Van Etten. Biochemical changes during the growth of fungi. I. Nitrogen compounds and carbohydrate changes in Penicillium atrovenetum. J. Bacteriol. 88:114-121. 1964.-Changes in the biochemical constituents of cells were studied during the growth and development of Penicillium atrovenetum. Growth of the fungus, as measured by the dry weight, could be divided into four phases: lag, log, stationary, and death. The percentages of total nitrogen, cold trichloroacetic acid-soluble nitrogen, ribonucleic acid (RNA), and protein increased to a maximum during the lag phase, and subsequently decreased as the fungus aged. The percentage of deoxyribonucleic acid (DNA) was always slightly higher in the spores than in the mycelium. The DNA in the mycelium decreased in the lag phase, and then increased slightly to a plateau for the duration of the log phase, followed by a decrease to a constant percentage during the stationary and death phases. Carbohydrates were present in higher concentration in the mycelium than in the spores. The percentage of carbohydrates in the mycelium increased continually until it reached a maximum late in the log phase, and then decreased as the fungus entered the death phase. The results reported for this fungus are, in general, in agreement with those reported for other microorganisms. Namely, the percentages of enzyme-forming compounds, such as amino acids, nucleotides, RNA, and protein, were highest in the lag phase, whereas storage compounds such as carbohydrates increased to a maximum near the end of the log phase. The definition of log phase in fungi depends on the criteria that are used. If, instead of using the linear increase in dry weight to delimit this growth period, one uses the end of net protein, RNA, and DNA synthesis, a more realistic concept of growth emerges.

Van Etten, James L. (University of Illinois, Urbana), and David Gottlieb. Biochemical changes during the growth of fungi. II. Ergosterol and fatty acids in Penicillium atrovenetum. J. Bacteriol. 89:409-414. 1965.-Changes in the lipid constituents of Penicillium atrovenetum were studied during the growth and development of this fungus. The stages in development, as measured by the dry weight, were divided into four phases: lag, log, stationary, and death. The total fatty acids on a dry-weight basis increased from a minimum in the spores to a maximum near the end of the log phase of growth. The major fatty acids were palmitic, stearic, oleic, and linoleic. Myristic, pentadecanoic, palmitoleic, heptadecanoic, linolenic, arachidic, and heptadecenoic acids, together with two unidentified components, were also present in the fatty acid fraction. Compared to ungerminated spores, young mycelium contained a much lower percentage (on the basis of total fatty acids) of linoleic acid. There was a corresponding increase of oleic acid. Except for palmitic acid, which remained constant, the remaining fatty acids increased slightly. During subsequent growth of the fungus, linleic acid decreased, whereas the percentage of palmitic and stearic acids increased steadily from the lag phase to the end of the log phase. Ergosterol was the only sterol detected. The percentage of ergosterol, on a dry-weight basis, increased to a maximum at the start of the log phase and then steadily decreased. The following changes in lipids appear to be associated with the development and aging of fungi: (i) the presence of a relatively high content of nonsaponifiable lipid and ergosterol in the young mycelium, and their later decrease with age, and (ii) a shift from more unsaturated to less unsaturated fatty acids with age.

Van Etten, James L. (University of Illinois, Urbana), H. Peter Molitoris, and David Gottlieb. Changes in fungi with age. II. Respiration and respiratory enzymes of Rhizoctonia solani and Sclerotium bataticola. J. Bacteriol. 91:169-175. 1966.-The rate of respiration of Rhizoctonia solani and Sclerotium bataticola decreased with age. This decrease in respiratory rate might be produced by a decrease in the specific activity of one or more enzymes involved in carbohydrate metabolism. Specific activities in cell-free extracts were measured for most of the enzymes in the hexose monophosphate shunt, Embden-Meyerhof-Parnas pathway, tricarboxylic acid cycle, and terminal electron-transport system. In addition, glucose oxidase, isocitritase, and malic enzyme were measured. In R. solani, increases in activity with age occurred for hexokinase, alpha-glycerolphosphate dehydrogenase, malic dehydrogenase, and cytochrome oxidase. Decreases occurred for phosphohexokinase, aconitase, nicotinamide adenine dinucleotide-specific isocitric dehydrogenase, reduced nicotinamide adenine dinucleotide oxidase, and at least one of the enzymes between 3-phosphoglycerate and pyruvate. In S. bataticola, increases in activity with age were observed for phosphohexokinase, pyruvic dehydrogenase, fumarase, malic dehydrogenase, and malic enzyme, whereas none of the enzymes decreased. The specific activities of the remaining enzymes did not change with age in either fungus.