Submerged cultures of four Streptomyces species accumulated triacylglycerol (TAG), ranging from 50 to 150 mg (I medium)-', during the post-exponential phase of growth. 5. lividans also produced glycogen (80 mg I l). Identity of TAG species was confirmed after TLC, by mass spectroscopy and by quantitative IR spectroscopy and reaction of the hydroxamate derivatives with ferric chloride. This is the first substantive report showing the storage of TAG in bacteria. Distribution of diacylglycerol acyltransferase (TAG synthase) activity from 5. coelicolor and 5. lividans during incubation on different media paralleled the formation of TAG. Accumulation of TAG may be necessary to maintain cell integrity after glucose becomes exhausted from the medium, and also to provide the C, units needed for subsequent biosynthesis of acetate-derived antibiotics in appropriate species. Actinorhodin was formed by 5. coelicolor when grown on YEME medium only after exhaustion of glucose; the carbon source may therefore originate from TAG. All the organisms examined in this study formed isoprenoid-derived hydrocarbons (up to 3 mg I ') which were identified as squalene plus hydrogenated derivatives.
Triacylglycerol is accumulated by Streptomyces spp. when grown in submerged culture. Ultrastructural studies using transmission electron microscopy (TEM), staining and freeze-fracture/freeze-etch procedures, and light microscopy confirmed the accumulation of neutral lipid by S. lividans and S. coelicolor during the stationary phase and its storage within membrane-bound globular structures within the cytoplasm. These structures were of various sizes and occupied up to approximately 80% of the total cell volume at that time. There was no evidence of such material within cells examined during the early exponential phase of growth. The globules visualised by TEM were electron-transparent since they comprised lipids containing saturated fatty acids that did not react with osmium tetroxide. The globules appeared to be bounded by a single membrane.
Investigations have been conducted into the relationship between fatty acid and phenol synthesis in submerged cultures of Aspergillus fumigatus. Both groups of metabolites are derived from acetate but phenol synthesis only occurs a t a late stage in the incubation period and is dependent on a change in the internal environment of the mycelium. Secretion of acetate-derived phenols commenced many hours after exhaustion of ammonium ions from the culture medium, or transfer of mycelium to glucose solution. Lipid synthesis, however, was maintained a t this stage and continued into the phenol-secreting phase when sufficient glucose was present to provide the necessary substrates. The additional lipid formed was identified as triglyceride.The composition of fatty acids in the total lipids was very similar to that found in other , Aspergillus sp. and related Ascomycetes and consisted principally of C18:2 with c16:0, c 1 6 : 1 , C1s:o and Cmi. Addition of 4-fluorophenylalanine, but not cycloheximide, caused a rapid reduction in fatty acid synthetase activity and lipid synthesis, and abolished formation of orsellinic acid and trihydroxytoluene, the normal secreted products.Certain enzymes examined, including the dehydrogenases of the pentose phosphate pathway, maintained their specific activity during the "stationary" phase whereas fatty acid synthetase was present at greatly reduced activity. The activity of ATP citrate lyase was severely reduced to an undetectable level after approximately 40 h incubation; acetyl-CoA would therefore not be made available for fatty acid, phenol or ergosterol synthesis after this time.Initiation of phenol synthesis cannot be ascribed simply to diversion of substrates from fatty acid synthesis or to reduction in fatty acid synthetase activity.Fatty acids together with a large group of naturally-occurring phenols are formed by means of condensation reactions between acetyl-CoA and malonylCoA. The synthesis of the latter products, however, entails few if any reductive steps and the oxygen functions from the malonyl residues are therefore often retained as phenolic groups. A phenomenon that is common to the synthesis of fatty acids and phenols in fungi lies in their formation as a response to deprivation of an essentialnutrient with a resultant disturbance to the internal environment of the cell [l]. Growth of the organism then ceases and the mycelium enters a stage that resembles the stationary Abbreviation. POPOP, p-bis,2-(5-phenyloxazolyl)benTrivial Name. Ergosterol, 5,7,22-ergostatrien-3B-01. Enzymes.
1. Orsellinic acid has been detected as a metabolite of Aspergillus fumigatus. 2. The other principal aromatic components of the medium are fumigatin and the quinol, fumigatol. Fumigatol has been shown to be dihydrofumigatin after oxidation to the quinone followed by acetylation. 3. (14)C-labelled 6-methylsalicylic acid can be hydroxylated in A. fumigatus to form orsellinic acid and decarboxylated to give m-cresol. 4. (14)C-labelled 6-methylsalicylic acid is incorporated into fumigatin and fumigatol (1.0-1.5%), but the conversion does not occur until about 2-3 days after supplementation of the medium. At this stage of growth, the organism has already synthesized approx. 20 times as much fumigatol as fumigatin and this ratio is reflected in the much lower specific activity of the quinol. 5. Supplementation of the medium with either orsellinic acid or orcinol, in addition to (14)C-labelled 6-methylsalicylic acid, greatly decreases the latter's incorporation into fumigatin. At the same time, the cultures containing these substances are stimulated to produce another quinone with relatively high specific activity. 6. 6-Methylsalicylic acid has not been detected in the medium of normal cultures. The results indicate that 6-methylsalicylic acid itself is not a direct precursor of fumigatin and fumigatol but that it is converted into a true intermediate, probably after hydroxylation to orsellinic acid. 7. Supplementation of the medium with 6-methylsalicylic acid (15-25mg./200ml.) greatly affects the metabolism of A. fumigatus. Growth is inhibited and the synthesis of fumigatol is markedly depressed in these cultures. The inhibitory effects may possibly be related in some way to the production of m-cresol.
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