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Control of fungal growth and metabolism
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CONTROL OF FUNGAL GROWTH AND METABOLISM The Physiology Group held a meeting on 'Control of Fungal Growth and Metabolism' at the School of Biological Sciences, University of Nottingham, on 22 - 24 September, 1980. There were fourteen invited papers at the meeting covering a broad range of topics. On the first day a session of open papers was held when eleven people contributed. The meeting was small in comparison to meetings held in London in recent years, however, most of the 75 people present stayed together in a hall of residence adjacent to the School of Biological Sciences. This aspect adds an important flavour to a meeting with many an interesting discussion taking place in the bar until a late hour. Once again we were pleased to welcome several participants from European countries, many of whom presented papers. Another feature of the Physiology Group meetings was also repeated namely the presence of a sizeable group of young participants. It is encouraging to see that the study of, and interest in, Mycology by younger scientists continues unabated. John F. Peberdy OFFERED PAPERS
Endogenous chitin synthase inhibitor in Aspergillus nidulans. G.D. Craig, J .McA. Campbell and J.F. Peberdy , Department of Botany, University of Nottingham, Nottingham, NG7 2 RD.
Chitin synthase exists in an active and a zymogen form in A. nidulans (Ryder, N.J. and Peberdy , 1.F., 1. gen. Microbial. (1977), 99,69). The zymogen component is activated in vitro by the action of trypsin and by an endogenous neutral protease which has been isolated from the mycelium of A. nidulans (Campbell, 1. MeA. and Peberdy, 1.F., FEMS Microbiol Lett. 6, 65). A glycoprotein chitin synthase inhibitor was isolated and partially purified from a cytoplasmic and membrane fraction of A. nidulans. The inhibitor was removed from the membrane fraction by treatment with protease. The inhibitor was heat-stable, non dialysable and a competitive inhibitor of active chitin synthase. Effects of polyene ant obio tics on chitin synthetase. D.M. Rast and R. Furter, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland and S. Bartnicki-Garcia, Department of Plant Pathology, University of California, Riverside, California 92521, USA.
The effect of amphotericin B, amphotericin B methyl ester, aureofungin, nystatin, candicidin, candidin, filipin, and pimaricin on purified chitin synthetase (chitosomes from yeast cells of Mucor ro ux ii ) was studied. These polyenes increased the activity of the enzyme by 10-60% at concentrations of 10-7 10 -5 M. Amphotericin B, its methyl ester, aureofunfin, nystatin and candicidin also inhibited it, the apparent K) values being 10- - 10-4 M; but filipin and pimaricin were not inhibitory. The polyenes that can inhibit chitin synthetase are members of the recently described group of polyenes displaying separation of fungistatic levels from fungicidal ones. Our findings indicate that sterol is essential for the normal functioning of chitin synthetase. The different response of the enzyme to various polyenes shows that the inhibitory effect of these compounds is not simply the result of action upon a non-specific sterol-binding site of the chitosomal membrane.
10 (3-glucan synthesis by membrane fraction of Saprolegnia. Michel Fevre, Labora-
toire de Physiologie Vegetale, Laboratoire de Mycologie assode au CNRS no 44, Universite Claude Bernard, 43 Boulevard du 11 Novembre 1918; 69622 Villeurbanne cedex, France. (3-g1ucan synthetase activity of the fungus Saprolegnia m ono ica was assayed by supplying UDP-Glucose to membrane fractions of mycelial homogenates. Analysis of glucan products by hydrolysis with various (3-g1ucanases and by chromatography shows that both (3 1-3 and {3 1-4 linkages are formed at high substrate concentration. In the absence of MgCI 2 , {3 1-3 linked glucans are mainly produced. The total synthesis activity and {3 1-3 linkaged production are reduced by increasing MgCl 2 concentrations. At low substrate concentration in the presence of MgC12 , (3 1-4 linked glucans are the only polysaccharides synthesized. Electron microscopy of products synthesized by the original membrane fraction or by membranes isolated from density gradients shows micro fibrils when assays are conducted in conditions to produce (3 1-3 glucans. Trypsin stimulates {3 1-3 glucan synthetase activity; on the contrary {3 1-4 glucan synthetases are inhibited by this protease. Comparison of trypsin and digitonin actions and trypsin stimulation of digitonin solubilized enzymes indicate that {3 1-3 glucan synthetases may exist in the cell under an inactive form (zymogen state). The ultrastructure of native and synthetic Agaricus bisporus melanins. D. Rast, H. Hegnauer and L.E. Nyhlen, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland.
The outer layer of the spore wall of A. bisporus containing chitin embedded in a matrix of (3-g1ucan-protein is heavily melanized. Electron microscopy of spore wall fragments that have been solvent extracted and enzymatically digested suggested that the melanin is present particularly in an amorphous state and partly as granules (cf. Rast and Hellenstein, Can. J. Bot. 55, 2251 (977)). Melanization starts at an early stage of wall formation and probably proceeds simultaneously with fibril deposition. Melanin obtained by exhaustive extraction of spores consists of two morphologically distinct components: (0 electrondense spheres of 50-100 nm in diameter and (ii) plate-like and less electrondense particles. Melanin synthesized in vitro from r-L-glutaminyl-4-hydroxybenzene (GHB; the natural phenolic precursor of the melanin of the A. bisporus spore wall) with crude mushroom phenol oxidase and with a stream of air passing through the mixture is similar to the native melanin of the spore wall, not only with respect to elemental composition, solubility properties and IR spectrum but also ultrastructural appearance: preparations of GHB melanin consist of round electron-dense bodies 30-200 nm in diameter embedded in amorphous material of medium electron density. The control of carbohydrate metabolism during periodic fruiting of Agaricus bisporus, J.B.W. Hammond, Glasshouse Crops Research Institute, Worthing Road, Rustington, Littlehampton, West Sussex, BNl6 3PU. Agaricus bisporus produces the majority of fruit bodies in periodic flushes.
The enzymes glucose-o-phosphate dehydrogenase (G6PD), mannitol dehydrogenase (MD) and glucose phosphate isomerase (GPO were assayed in fruit bodies at two stages of development, produced during flushes and between flushes. G6PD increased markedly in fruit bodies at both stages of development
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to a peak between flushes. MD followed this pattern, but did not show such a great variation in activity. GPI showed greater activity between flushes in young fruit bodies but peaked during the flush in older fruit bodies. It is suggested that the rise in G6PD activity is initiated by a stimulus from the mycelium , while GPI activity is controlled by the fruit body. The possible significance of the changes in enzyme activity in the control of mannitol metabolism and flush growth will be discussed. The effect of glucose on the potential of sclerotial production in submerged liquid culture of Sclerotium rolfsii. Y. Hadar , I. Chet and Y. Henis, Department of Plant Pathology and Microbiology , Faculty of Agriculture , The Hebrew Univers ity of Jerusalem , Rehovot , Israel. Sclerotium rolfsii grown in a shaken submerged culture did not produce any sclerotia. When 15 ml aliquots of the shaken culture of S. rolfsii , grown in medium containing 1.5% glucose , were poured into Petri dishes and incubated for 16 h their surface became exclusively covered with vegetative mycelium, if poured within the first 40 h of shaking. However when the aliquots were poured after 50 h of shaking only sclerotial initials were formed. This phenomenon occurred simultaneously with glucose exhaustion and when fungal biomass reached its maximal level. When glucose concentration was increased to 4% the potential of sclerotia production appeared after 120 h of shaking, the period required for glucose exhaustion. This pattern was maintained at glucose con centrations between 0.5 % and 4%. When grown in continuous culture with glucose at limiting concentration (0.2%) at a dilution rate of 0.05 h- I aliquots poured into Petri dishes produced sclerotial initials within 12-16 h. At a non-limiting glucose concentration (0 .5%) poured samples developed mycelium only. It is suggested that sclerotial production in S. rolfsii is an endogenous process controlled by external nutrients. Environm ental control of developm ent in Penicillium claviforme : effect of differing rates of amino acid assimilation. Sarah C. Watkinson, Botany School, University of Oxford, South Parks Road, Oxford, OXI 3RA.
Glutamate concentration controls numbers of coremia formed, and their earliest stages can be identified within a few hours of spore germination , making them attractive subjects for investigation of the mechanism of an external stimulus to multihyphal development. Uptake and incorporation of serine and glutamate were measured in surface grown mycelium at varied medium concentrations, during th e time that coremium primordia were forming . Both amino acids supported the same amount of vegetative growth but both uptake and incorporation of 14 C-glutamate were faster and more closely related to medium concentration than uptake and incorporation of labelled serine . Medium glutamate made a larger contribution to the internal amino acid pool than did serine. The morphogenetic effect of glutamate may result from its ability to support adequate biosynthesis for development during the limited period when the mycelium is able to form coremia. Control of phosphate uptake in Neurospora crassa. R.E. Beever* and D.J.W. Burns , Plant Diseases Division, Department of Scientific and Industrial Research, Auckland , New Zealand , ( *temporary address: Long Ashton Research Station, Long Ashton , Bristol , BS18 9AF).
Phosphate uptake in Ne urospora crassa is due to the act ivity of two systems , one of high -affinity (Km 2.75mM) and one of low-affinity (Km varies between 370 - 1029 11M) . Uptake into germlings growing exponentially at phosphate
12 concentrations between 50 J.LM and 10 mM is constant. The manner in which the Km and Vmax of the uptake systems alter to achieve this constancy has been explored in detail. The mechanisms controlling the systems have been investigated in cultures transferred to either higher or lower phosphate concentrations. The high-affinity system is depressed under conditions of phosphate starvation, and inhibited irreversibly by feedback inhibition under conditions of oversupply. The low-affinity system is also derepressed and subject to feedback inhibition under comparable conditions, but, in contrast, inhibition of this system is reversible. Growth of Aspergillus flavus on p-coumaryl alcohol. C.B. Iyayi and R.K. Dart, Microbiology Section, Department of Chemistry, University of Technology, Loughborough, Leics, LEI 1 3TU. Aspergillus flavus has been shown to grow on p-eoumaryl alcohol which is one of the major constituents of lignin. Labelling experiments have been carried out using C 14 -p-coumaryl alcohol, and p-eoumaric acid, acetic acid, p-hydroxybenzoic acid, protocatechuic acid and {3 -keto-adipic acid have been isolated. Identification of these compounds has included melting point determinations, I.R., N.M.R. and mass spectrometry. A pathway for the breakdown of p-coumaryl alcohol is proposed. Rapid detection and estimation of spores of Aspergillus flavus. J .0. Offem and R.K. Dart, Microbiology Section, Department of Chemistry, University of Technology, Loughborough, Leics, LE 11 3TU. Aspergillus flavus is a contaminant of many crops in Nigeria causing considerable spoilage and serious economic losses due to the production of aflatoxin. Two methods have been developed for the detection of spores of A. flavus which allow estimation of spore numbers within 18 h. Both methods are based on the ability of the fungus to hydrolyse the ester linkages of pectin. Chlorate resistance and nitrogen assimilation in industrial strains of Penicillium chrysogenum. J .A. Birkett and R.T. Rowlands, Glaxo Operations UK Ltd., Ulverston, Cumbria LA 12 9DR. England.
Several chlorate resistant mutants of Penicillium chrysogenum have been isolated and analysed, all were affected in nitrate assimilation. Nine loci have been recognised by complementation analysis and these appear to be equivalent to the niaD (nitrate reductase structural gene), nirA (control locus) and seven cnx loci (responsible for the biosynthesis of a cofactor for nitrate reductase) of Aspergillus nidulans. The organisation of the nitrate assimilation genes appears to be similar in both organisms even to the extent of having continguous genes coding for the nitrate and nitrite reductase enzymes. INVITED PAPERS
Chemostat control of hyphal macromolecular content. M.E. Bushell, Department of Microbiology, University of Surrey, Guildford, Surrey, GU2 5XH.
Studies of the effects of growth rate on the macromolecular composition of microorganisms were facilitated when reliable methods of chemostasis were developed. Experiments were prokaryotes have shown that, the proportions of DNA and protein are independent of growth limitation and remain constant over a range of growth rates. RNA content decreases with doubling time. Carbon-rich storage materials, polysaccharides and lipids, are more abundant at low growth rates, particularly under nitrogen limitation.
13 Data on individual macromolecules from different filamentous fungi appear contradictory (e.g. Griffen, D.H. et al, J. gen Microbiol. (1974), 80, 381; Bu'Lock, J.D. et aI, Trans. Br. mycol, Soc. (1974), 62,377). When such results are considered in the context of specific cellular functions, however, some uniformity in the effects of growth rate on hyphal macromolecular content becomes apparent. An expression for ribosomal efficiency has been proposed (Alroy, Y. and Tannenbaum, S.R., Biotech, Bioeng. (1973), 15,239) involving the ratios of protein, RNA and DNA productivity in terms of the relative specific growth rate. Subsequent calculations by the author have revealed linear relationships between ribosomal efficiency and growth rate per se, The appropriate plots passed through the origin in prokaryotes and cut the abscissa at the minimum growth rate for purely vegetative growth in fungal cultures capable of differentiation. This observation therefore, supports the concept of a minimum specific growth rate (Pirt, S.J., Appl. Chem. Biotech. (1972),22, 55). Studies with different species on the variation of hyphal lipid and carbohydrate content also appear contradictory when considered in isolation. In Aspergillus nidulans no simple relationship between intracellular concentration and doubling time was apparent for either polymer. When the combined energy storage equivalent value (Forrest, W.W. and Walker, DJ. (Adv. Microb. Phys. (I971), 5, 213) was calculated however, a linear relationship between energy value and growth rate was established. Growth related changes in ribosomal efficiency and DNA productivity may be related to corresponding alterations in hyphal polyamime content. Attempts to deduce ad hoc relationships between polyamine content and fungal morphogenises have been unsuccessful (Marshall, M. et al, Curro Microbiol. (I979) 2, 187). Evidence for a more fundamental connection between polyamines and fungal growth will be presented. Factors affecting the growth of dimorphic fungi. F.W. Chattaway , Department of Biochemistry, University of Leeds, Leeds, LS2 9LS. The control of morphological development in dimorphic fungi is of intrinsic interest in relation to the role of environmental factors and of probable interest in the pathogenicity of some species in mammalian hosts. The cell wall may have a role in determining morphology and differences have been shown in the composition of cell wall from yeast and mycelial forms in terms of glucan, chitin and protein content. Environmental factors which can affect morphological development are temperature, pH, oxygen supply, glucose concentration and the nature of the nitrogen source in the medium; in addition the size of the inoculum and the age of cells used can be modifying factors. Biochemical systems which have been studied as possibly affecting the observed type of growth are (i) chitin synthetase in Candida albicans, (ii) glycolysis and related pathways in Mucor spp. and Candida albicans, (iii) protein synthesis in relation to sulphur metabolism and RNA polymerases in Histoplasma capsula tum . Biochemical activity in many organisms is regulated by cyclic -3', 5' -adenosinemonophosphate, and there is increasing evidence for its possible role in controlling dimorphism. The autolytic phase of growth in filamentous fungi: shape and size of vacuoles of Neurospora crassa. R. Lehoz and F. Reyes, Instituto 'Jaime Ferran' de Microbiologia del e.S.I.C., Joaquin Costa, 32, Madrid-6, Spain. Changes in the shape and size of vacuoles of Neurospora crassa, as seen under the light microscope, in relation to autolysis have been studied. Of a total hyphal length amounting to 22,600 11m 2,970 vacuoles were drawn, counted and
14 measured during pre-autolysis and autolysis, being 66.3% apparently circular or very nearly circular shaped. In the period between the first 24-36 hours incubation the mean vacuolar radius underwent an increase in length amounting to 44.4%. As incubation proceeded a lateral elongation of most of these circular vacuoles seems to take place. At the end of the log phase the ratio circular/non-circular vacuoles amounted to 8.1: 1; during the pre-autolytic stages this ratio decreased to 2.4: 1, being 0.4: 1 at the end of autolysis. Plotting the values of this ratio against time of incubation it yielded a hyperbolic curve. Measuring vacuolation as the change in the area of vacuoles, it has been observed that from a total hyphal area of 131,534 IJ.m 2 (measured in 369 hyphae) the vacuolar area increased nearly 25% during the pre-autolytic period, and 30% during autolysis, therefore a continuous increase of this vacuolar area with time of incubation occurred.
Control of growth and development by volatile metabolites. P.M. Robinson, Department of Botany, The Queen's University, Belfast, BT7 1NN. All stages of development of Geotrichum candidum can be influenced by selfproduced volatile metabolites. Arthrospores fail to germinate when seeded at a concentration of ~ lOs arthrospores ml- 1 in distilled water or distilledwater agar and this self-inhibition of germination is due to the production of volatile metabolites which retard germination when present at low concentrations. The increase in concentration of these metabolites which occurs with increasing arthrospore concentration parallels the increase in the level of selfinhibition of the arthrospore population and the associated decrease in negative autotropism of arthrospore pairs. The autotropic response may be due to or influenced by the volatile metabolite(s) responsible for self-inhibition. Hyphal development in G. candidum is affected by volatile metabolites produced by germinated or ungerminated arthrospores. Arthrospores exposed to these metabolites produce hyphae which sporulate earlier than untreated hyphae. At high concentrations of these metabolites hyphal extension is arrested abruptly and arthrospore formation does not occur; a decrease in the concentration of these metabolites results in continued extension-growth of the hyphae. The metabolites responsible for the affects described have not been characterized but preliminary work has established that the effects are not due to oxygen deficiency or to the production (or absence) of carbon dioxide.
Growth rate and metabolic control in yeast. D.R. Berry, Department of Applied Microbiology, University of Strathclyde, George Street, Glasgow, GI lXW. It is well established that the levels of carbon, nitrogen and phosphorus in the growth medium markedly influence the growth and development of microorganisms. In yeast the role of glucose in catabolite repression is well established and nitrogen depletion has been reported to influence glycogen storage. The levels of carbon, nitrogen and phosphorus are also known to be important in controlling the quality of bakers yeast. In this paper the effect of variations in the level of glucose, ammonium and phosphate on the metabolism of yeast have been studied. Techniques have been developed for the rapid and accurate analysis of intermediates of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and the glyoxylate cycle in yeast grown in batch and continuous culture. The application of the techniques to batch culture gave complex results.
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The use of continuous culture provided stable , precisely defined physiological conditions for studying the level of intermediates. The results indicated that intermediate levels in the pathways studied are a function of the nutritional status of the medium and of the growth rate. They appeared to be stable for a given steady state , however fluctuations in the levels of intermediates could be induced by the add ition of nutrients in short pulses .
Carbohydrate metabolism and morphogenesis. D. Moore, Department of Botany, University of Manchester, Manchester, Ml3 9PL. The varied relationship between carbohydrate metabolism and fungal morphogenesis will be illustrated by discussion of a number of different examples. The part played by enzymes responsible for interconversion of sugars is clearly important , since mutations in genes specifying these enzymes often lead to morphological abnormalities of hyphal and vegetative colony growth. Similar abnormalities can also be induced by inhibitors and experiments with the latter represent a means of studying the way in which hyphal form can be modified. During normal morphogenesis changes which represent steps in development are precipitated by both environmental and metabolic events. Agents which might be involved in signalling such changes include the ubiquitous cyclicAMP, but may also include metabolic intermediates. This point will be illustrated by discuss ion of the possible role of pyruvate in the regulation of glutamate dehydrogenase during vegetative growth of a strain of Coprinus cinereus in relation to events which occur endogenously during morphogenesis of the carpophore cap.
Genetic approaches to the control of nitrogen metabolism in Aspergillus nidulans . H.N . Arst , Jr ., Department of Genetics, University of Newcastle upon Tyne , Newcastle upon Tyne, NEI 7RU . The syntheses of many enzymes and permeases which playa role in the nitrogen nutrition of Aspergillus nidulans are subject to one or more forms of regulation . These include induction, nitrogen metabolite (i.e. ammonium) repression, and, where there is an involvement in carbon nutrition, carbon catabolite repression. All of the regulatory genes mediating induction investigated to date are positive acting and can be classified as pathway-specific or integrator (i.e . mediating concomitant induction of activities coded by two or more non-contiguous structural genes of which at least one can also be expressed independently by an alternative induction mechanism). Nitrogen metabolite repression is mediated by a positive acting regulatory gene designated areA , and ammonium (or a metabolite derived from it) inactivates (or less likely represses formation of) the are A product. In at least some cases carbon catabolite repression is mediated by a negatively acting regulatory gene designated creA. Cis-acting receptor sites for these regulatory gene products have been identified by mutations in a few cases. In one case a new regulatory pattern has arisen from fusion of a structural gene to a promoter normally located in a different linkage group as the result of an insertional translocation. As in other eukaryotes , functionally related genes are seldom clustered in A . nidulans. Some exceptions to thi s generalisation include 'cluster-genes' coding for polyfunctional polypeptides and a few bona fide gene clusters whose significance and genetic organisation is under study. In one such cluster, there is genetic evidence for synthesis of a dicistronic transcript plus at least one (mono-, tri- , or tetracistronic) overlapping transcript .
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Biosynthesis of amino acids in yeasts and its exclusion from degradation . J-M. Wiame, Laboratoire de Microbiologie, Universite Libre de Bruxelles, et Institut de Recherches du CERIA, 1 avo E. Gryson, B-1070 Brussels, Belgium . Yeasts have the simultaneous possibility of synthesis and degradation of aminoacids. Occasionally some intermediary metabolites are common to both processes and bring up the problem of futile cycle exclusion . Experimental production of such a cycle by mutation has allowed us to appreciate the factors which avoid the occurrence of this waste in normal cells . In asparagine synthesis the exclusion is remarkably simple and stands on an adequate adjustment of enzyme kinetic parameters. The comparative study of a number of yeast genera illustrates the possibility of appreciating the physiological factors which may have been involved in the selection of two types of futile cycle avoidance. In arginine metabolism , the occurrence of an epiarginasic regulation versus a pro cess of compartmentation involving the mitochondrion appears to be correlative with energy metabolism . In addition to enzymes as the direct target, exclusion at the level of an enzyme synthesis is also illustrated in arginine metabolism at the level of genetic regulatory circuits. In that case a repressor ARGR has two distinct functions (ambivalent repressor): it represses as usually the biosynthetic enzymes in binding with operators and, in addition, it removes the catabolic specific repressor CARG R from the operators of the two genes coding for catabolic enzymes. The dual function of ARGR is nicely shown by a unique mutation which favours catabolism (and is dominant over wild type) and simultaneously weakens its anabolic role and is recessive.
Control of chitin synthesis. G.W. Gooday , Department of Microbiology , Marischal College, University of Aberdeen, Aberdeen, AB9 1AS. The activ ity of the enzyme chitin synthase is affected by a range of metabolites and cell constituents. These include uridine-containing molecules , especially the substrate UDP-N-acetylglucosamine and the product uridine diphosphate; the monomer and dimer of chitin , N-acetylglucosamine and N ,N '-
Control of lipid metabolism in y east and fungal growth . Anthony H. Rose, Zymology Laboratory, School of Biological Sciences, University of Bath, Bath, Avon . Yeast and fungal lipids are made up of two classes. The polar lipids include phospholipids and sterols, which are located in the plasma membrane with intracellular membranes. The non-polar lipids, which include triacylglycerols and sterol esters, are located not in membranes but in intracellular low-density vesicles . Overproduction of phospholipids and sterols is alm ost ne ver observed, but many yeasts and fungi are able to produce large amounts of neutral lipids, so metimes accounting for as much as 60% of the dry weight of the biomass. Metabolic pathways have been charted for synthesis of all of the major classes of yeast and fungal lipids , and there is evidence that the amounts synthesized are regulated by well established feedback processes. However , many problems remain unsolved in the biosynthesis of yeast and fungal lipids . The two major phospholipids , namely phosphatidylcholine and phosphatidylethanolamine, can
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be synthesized by either the cytidine nucleotide or the methylation pathway. Little is known of the metabolic factors which dictate which of the pathways is used for each of these phospholipids. Nor is there more than a cursory understanding of the mechanisms which regulate the fatty-acyl composition of phospholipids , triacylglycerols and sterol esters. While the subcellular location of the various classes of lipid has been established , that of the sites in the cell where the various lipids are synthesized is unclear. Finally , while there is evidence for a role for neutral lipids in biosynthesis of polar lipids , information on this role is meagre .
Some relationships between primary and secondary metabolism . M.O. Moss, Department of Microbiology, University of Surrey, Guildford, Surrey, GU2 5XH. Several decades of intensive study of the chemistry of secondary metabolites have uncovered a considerable diversity of structures. At the biochemical level, it has been possible to see patterns in this diversity through the elucidation of biosynthetic pathways and the demonstration that relatively few fundamental pathways are involved in the production of thes e metabolites. It is at the biosynthetic level that the links between primary and secondary metabolites are most apparent for the biosynthesis of each involves the same relatively few low molecular weight precursor molecules . Thus acetyl coenzyme A is a precursor of both fatty acids and polyketides , amino acids are precursors of both proteins and diketopiperazines, [3-lactams and alkaloids, whereas mevalonate is a pre cursor of terpenes, sesquiterpenes and diterpenes as well as sterols and carotenoids. At the physiological level, the relationship between primary and secondary metabolism as time dependent processes is less certain. Any general theory of secondary metabolism must take account of the very large number of chemically distinct, and apparently genetically defined, compounds described as secondary metabolites as well as the specificity with which they are frequently associated with individual species of microorganism. It must also take account of the physiological state of the producing organism at the time of optimum pro duction of secondary metabolites , as well as the often whimsical nature of the production of particular compounds by some species. The pharmaceutical industry has adequately demonstrated that it is possible to manipulate both the genetic and physiological status of a species to optimise , and often very dramati cally increase, the yield of a particular metabolite with useful pharmaceutical properties . Bearing these points in mind, this paper will consider some speculative aspects of the natural history of primary and secondary metabolism.
Genetic control of penicillin produ ction. K.D. Macdonald , Chemical Defence Establishment , Porton Down , Salisbury , Wilts, SP4 OJQ . Penicillin is elaborated by a number of fungi including Aspergillus nidulans and Penicillium chrysogenum . The latter is employed for the industrial production of penicillin and the possibility of using the parasexual mechanism it possesses to breed improved strains has been explored. Aspergillus nidulans , which has both sexual and parasexual cycles , yields much less penicillin than Penicillium chryso genu m but because its formal genetics is much better understood it is a useful model organism for genetic studies on penicillin production. An examination of mutants of Penicillium chrysogenum impaired in penicillin biosynt hesis established that there were five different lo ci involved and a similar number of loci were found in Aspergillus nidulans. Studies with mutants of Penicillium chrysogenum which overproduced penicillin indicated that these were usually recessive. When attempts were made
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to cross mutants from divergent lines it was found that most of the progeny had one or other parental genome probably due to different chromosomal rearrangements in the parental strains. The lack of recombination and thus an inability to isolate progeny with better penicillin yields than either parent was overcome by avoiding mutagens causing gross chromosomal damage and crossing strains not widely divergent in lineage. Three mutants of Aspergillus nidulans with increased penicillin yields were positioned on three different linkage groups . Crosses indicated that one was recessive , one semi-dominant and the third , dominant. The recessive mutant was epistatic to the other two , and the semi-dominant mutant epistatic to the dominant mutant. Crosses between different wild type strains of As pergillus nidulans yielded progeny with better penicillin yields than either parent.
Physiological aspects of my cotoxin production in f ermenter conditions. I.E. Smith , Department of Applied Microbiology, University of Strathclyde, George Street, Glasgow, G I I XW. Aflatoxins are a group of related difunaro-coumarin secondary metabolites which are produced by Aspergillus [lavus and Aspergillus parasitic us. These mycotoxins are acutely toxic and have been shown to be carcinogenic to certain anim al species. Aflatoxins normally enter the dietary chain by way of agricultural products contaminated by toxin producing moulds. Much interest has been con cerned with the effect of various acids , namely propionic acid , on controlling the growth of these organisms and toxin production . The present paper will attempt to study some aspects of aflatoxin production by Aspergillus flavus in liquid and solid state fermentation conditions. In particular, these results will show propionic acid at certain concentrations promotes a stimulation of aflatoxin production by the producer organism. These effe cts have been studied in liquid and in solid surface cultivation. In particular, these studies show that the stimulated effect of propionic acid occurs during the early hours of spore germination. Propionic acid has no effect on an actively growing vegetative culture.
Control of ergot alkaloid biosynthesis. P.G . Mantle , Department of Biochemistry , Imperial College , London , SW7 2AY. Ergot fungi almost invariably produce their characteristic alkaloids while growing as plant parasites but the yields are generally less than 0.5%. In some species clavine alkaloids are produced apparently wit hout any particular differentiation of a special growth form . Alkaloid production in submerged cultur e is therefore fairly readily achieved provided that glucan accumulation is controlled . In other species, noted for their ability to elaborate the more complex alkaloids (the lysergic acid derivatives), the parasitic sclerotial tissue is quite different from any other tissue formed by the fungus. In Clav iceps purpurea , for example, sclerotial cells contain a triglyceride oil which is not produced by any other micro-organism and alkaloid is only biosynthesised by these lipid-rich cells . The control of sclerotial-like growth in axenic culture is a prerequisite for alkaloid production. Certain isolates of C. paspali have been manipulated in submerged culture to over-produce a lysergic acid isomer, or simple derivatives, by a factor of about 10" but this may reflect suppression of pathways normally diverting tryptophan into other secondary metabolites . Alkaloid production by C. purpurea in axenic culture is rare and the direct fermentation production of therapeutically-valuable alkaloids by this organism presents the principal challenge concerning understanding and applying control of alkaloid biosyntheses.
19 The main pathways of peptide alkaloid biosynthesis will be discussed and the extent to which enzyme systems will accept foreign precursor analogues will be illustrated by describing the bioproduction of a new cyclic tripeptide alkaloid analogue.
CONTROL OF FUNGAL GROWTH AND METABOLISM The Physiology Group held a meeting on 'Control of Fungal Growth and Metabolism' at the School of Biological Sciences, University of Nottingham, on 22 - 24 September, 1980. There were fourteen invited papers at the meeting covering a broad range of topics. On the first day a session of open papers was held when eleven people contributed. The meeting was small in comparison to meetings held in London in recent years, however, most of the 75 people present stayed together in a hall of residence adjacent to the School of Biological Sciences. This aspect adds an important flavour to a meeting with many an interesting discussion taking place in the bar until a late hour. Once again we were pleased to welcome several participants from European countries, many of whom presented papers. Another feature of the Physiology Group meetings was also repeated namely the presence of a sizeable group of young participants. It is encouraging to see that the study of, and interest in, Mycology by younger scientists continues unabated. John F. Peberdy OFFERED PAPERS
Endogenous chitin synthase inhibitor in Aspergillus nidulans. G.D. Craig, J .McA. Campbell and J.F. Peberdy , Department of Botany, University of Nottingham, Nottingham, NG7 2 RD.
Chitin synthase exists in an active and a zymogen form in A. nidulans (Ryder, N.J. and Peberdy , 1.F., 1. gen. Microbial. (1977), 99,69). The zymogen component is activated in vitro by the action of trypsin and by an endogenous neutral protease which has been isolated from the mycelium of A. nidulans (Campbell, 1. MeA. and Peberdy, 1.F., FEMS Microbiol Lett. 6, 65). A glycoprotein chitin synthase inhibitor was isolated and partially purified from a cytoplasmic and membrane fraction of A. nidulans. The inhibitor was removed from the membrane fraction by treatment with protease. The inhibitor was heat-stable, non dialysable and a competitive inhibitor of active chitin synthase. Effects of polyene ant obio tics on chitin synthetase. D.M. Rast and R. Furter, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland and S. Bartnicki-Garcia, Department of Plant Pathology, University of California, Riverside, California 92521, USA.
The effect of amphotericin B, amphotericin B methyl ester, aureofungin, nystatin, candicidin, candidin, filipin, and pimaricin on purified chitin synthetase (chitosomes from yeast cells of Mucor ro ux ii ) was studied. These polyenes increased the activity of the enzyme by 10-60% at concentrations of 10-7 10 -5 M. Amphotericin B, its methyl ester, aureofunfin, nystatin and candicidin also inhibited it, the apparent K) values being 10- - 10-4 M; but filipin and pimaricin were not inhibitory. The polyenes that can inhibit chitin synthetase are members of the recently described group of polyenes displaying separation of fungistatic levels from fungicidal ones. Our findings indicate that sterol is essential for the normal functioning of chitin synthetase. The different response of the enzyme to various polyenes shows that the inhibitory effect of these compounds is not simply the result of action upon a non-specific sterol-binding site of the chitosomal membrane.
10 (3-glucan synthesis by membrane fraction of Saprolegnia. Michel Fevre, Labora-
toire de Physiologie Vegetale, Laboratoire de Mycologie assode au CNRS no 44, Universite Claude Bernard, 43 Boulevard du 11 Novembre 1918; 69622 Villeurbanne cedex, France. (3-g1ucan synthetase activity of the fungus Saprolegnia m ono ica was assayed by supplying UDP-Glucose to membrane fractions of mycelial homogenates. Analysis of glucan products by hydrolysis with various (3-g1ucanases and by chromatography shows that both (3 1-3 and {3 1-4 linkages are formed at high substrate concentration. In the absence of MgCI 2 , {3 1-3 linked glucans are mainly produced. The total synthesis activity and {3 1-3 linkaged production are reduced by increasing MgCl 2 concentrations. At low substrate concentration in the presence of MgC12 , (3 1-4 linked glucans are the only polysaccharides synthesized. Electron microscopy of products synthesized by the original membrane fraction or by membranes isolated from density gradients shows micro fibrils when assays are conducted in conditions to produce (3 1-3 glucans. Trypsin stimulates {3 1-3 glucan synthetase activity; on the contrary {3 1-4 glucan synthetases are inhibited by this protease. Comparison of trypsin and digitonin actions and trypsin stimulation of digitonin solubilized enzymes indicate that {3 1-3 glucan synthetases may exist in the cell under an inactive form (zymogen state). The ultrastructure of native and synthetic Agaricus bisporus melanins. D. Rast, H. Hegnauer and L.E. Nyhlen, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland.
The outer layer of the spore wall of A. bisporus containing chitin embedded in a matrix of (3-g1ucan-protein is heavily melanized. Electron microscopy of spore wall fragments that have been solvent extracted and enzymatically digested suggested that the melanin is present particularly in an amorphous state and partly as granules (cf. Rast and Hellenstein, Can. J. Bot. 55, 2251 (977)). Melanization starts at an early stage of wall formation and probably proceeds simultaneously with fibril deposition. Melanin obtained by exhaustive extraction of spores consists of two morphologically distinct components: (0 electrondense spheres of 50-100 nm in diameter and (ii) plate-like and less electrondense particles. Melanin synthesized in vitro from r-L-glutaminyl-4-hydroxybenzene (GHB; the natural phenolic precursor of the melanin of the A. bisporus spore wall) with crude mushroom phenol oxidase and with a stream of air passing through the mixture is similar to the native melanin of the spore wall, not only with respect to elemental composition, solubility properties and IR spectrum but also ultrastructural appearance: preparations of GHB melanin consist of round electron-dense bodies 30-200 nm in diameter embedded in amorphous material of medium electron density. The control of carbohydrate metabolism during periodic fruiting of Agaricus bisporus, J.B.W. Hammond, Glasshouse Crops Research Institute, Worthing Road, Rustington, Littlehampton, West Sussex, BNl6 3PU. Agaricus bisporus produces the majority of fruit bodies in periodic flushes.
The enzymes glucose-o-phosphate dehydrogenase (G6PD), mannitol dehydrogenase (MD) and glucose phosphate isomerase (GPO were assayed in fruit bodies at two stages of development, produced during flushes and between flushes. G6PD increased markedly in fruit bodies at both stages of development
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to a peak between flushes. MD followed this pattern, but did not show such a great variation in activity. GPI showed greater activity between flushes in young fruit bodies but peaked during the flush in older fruit bodies. It is suggested that the rise in G6PD activity is initiated by a stimulus from the mycelium , while GPI activity is controlled by the fruit body. The possible significance of the changes in enzyme activity in the control of mannitol metabolism and flush growth will be discussed. The effect of glucose on the potential of sclerotial production in submerged liquid culture of Sclerotium rolfsii. Y. Hadar , I. Chet and Y. Henis, Department of Plant Pathology and Microbiology , Faculty of Agriculture , The Hebrew Univers ity of Jerusalem , Rehovot , Israel. Sclerotium rolfsii grown in a shaken submerged culture did not produce any sclerotia. When 15 ml aliquots of the shaken culture of S. rolfsii , grown in medium containing 1.5% glucose , were poured into Petri dishes and incubated for 16 h their surface became exclusively covered with vegetative mycelium, if poured within the first 40 h of shaking. However when the aliquots were poured after 50 h of shaking only sclerotial initials were formed. This phenomenon occurred simultaneously with glucose exhaustion and when fungal biomass reached its maximal level. When glucose concentration was increased to 4% the potential of sclerotia production appeared after 120 h of shaking, the period required for glucose exhaustion. This pattern was maintained at glucose con centrations between 0.5 % and 4%. When grown in continuous culture with glucose at limiting concentration (0.2%) at a dilution rate of 0.05 h- I aliquots poured into Petri dishes produced sclerotial initials within 12-16 h. At a non-limiting glucose concentration (0 .5%) poured samples developed mycelium only. It is suggested that sclerotial production in S. rolfsii is an endogenous process controlled by external nutrients. Environm ental control of developm ent in Penicillium claviforme : effect of differing rates of amino acid assimilation. Sarah C. Watkinson, Botany School, University of Oxford, South Parks Road, Oxford, OXI 3RA.
Glutamate concentration controls numbers of coremia formed, and their earliest stages can be identified within a few hours of spore germination , making them attractive subjects for investigation of the mechanism of an external stimulus to multihyphal development. Uptake and incorporation of serine and glutamate were measured in surface grown mycelium at varied medium concentrations, during th e time that coremium primordia were forming . Both amino acids supported the same amount of vegetative growth but both uptake and incorporation of 14 C-glutamate were faster and more closely related to medium concentration than uptake and incorporation of labelled serine . Medium glutamate made a larger contribution to the internal amino acid pool than did serine. The morphogenetic effect of glutamate may result from its ability to support adequate biosynthesis for development during the limited period when the mycelium is able to form coremia. Control of phosphate uptake in Neurospora crassa. R.E. Beever* and D.J.W. Burns , Plant Diseases Division, Department of Scientific and Industrial Research, Auckland , New Zealand , ( *temporary address: Long Ashton Research Station, Long Ashton , Bristol , BS18 9AF).
Phosphate uptake in Ne urospora crassa is due to the act ivity of two systems , one of high -affinity (Km 2.75mM) and one of low-affinity (Km varies between 370 - 1029 11M) . Uptake into germlings growing exponentially at phosphate
12 concentrations between 50 J.LM and 10 mM is constant. The manner in which the Km and Vmax of the uptake systems alter to achieve this constancy has been explored in detail. The mechanisms controlling the systems have been investigated in cultures transferred to either higher or lower phosphate concentrations. The high-affinity system is depressed under conditions of phosphate starvation, and inhibited irreversibly by feedback inhibition under conditions of oversupply. The low-affinity system is also derepressed and subject to feedback inhibition under comparable conditions, but, in contrast, inhibition of this system is reversible. Growth of Aspergillus flavus on p-coumaryl alcohol. C.B. Iyayi and R.K. Dart, Microbiology Section, Department of Chemistry, University of Technology, Loughborough, Leics, LEI 1 3TU. Aspergillus flavus has been shown to grow on p-eoumaryl alcohol which is one of the major constituents of lignin. Labelling experiments have been carried out using C 14 -p-coumaryl alcohol, and p-eoumaric acid, acetic acid, p-hydroxybenzoic acid, protocatechuic acid and {3 -keto-adipic acid have been isolated. Identification of these compounds has included melting point determinations, I.R., N.M.R. and mass spectrometry. A pathway for the breakdown of p-coumaryl alcohol is proposed. Rapid detection and estimation of spores of Aspergillus flavus. J .0. Offem and R.K. Dart, Microbiology Section, Department of Chemistry, University of Technology, Loughborough, Leics, LE 11 3TU. Aspergillus flavus is a contaminant of many crops in Nigeria causing considerable spoilage and serious economic losses due to the production of aflatoxin. Two methods have been developed for the detection of spores of A. flavus which allow estimation of spore numbers within 18 h. Both methods are based on the ability of the fungus to hydrolyse the ester linkages of pectin. Chlorate resistance and nitrogen assimilation in industrial strains of Penicillium chrysogenum. J .A. Birkett and R.T. Rowlands, Glaxo Operations UK Ltd., Ulverston, Cumbria LA 12 9DR. England.
Several chlorate resistant mutants of Penicillium chrysogenum have been isolated and analysed, all were affected in nitrate assimilation. Nine loci have been recognised by complementation analysis and these appear to be equivalent to the niaD (nitrate reductase structural gene), nirA (control locus) and seven cnx loci (responsible for the biosynthesis of a cofactor for nitrate reductase) of Aspergillus nidulans. The organisation of the nitrate assimilation genes appears to be similar in both organisms even to the extent of having continguous genes coding for the nitrate and nitrite reductase enzymes. INVITED PAPERS
Chemostat control of hyphal macromolecular content. M.E. Bushell, Department of Microbiology, University of Surrey, Guildford, Surrey, GU2 5XH.
Studies of the effects of growth rate on the macromolecular composition of microorganisms were facilitated when reliable methods of chemostasis were developed. Experiments were prokaryotes have shown that, the proportions of DNA and protein are independent of growth limitation and remain constant over a range of growth rates. RNA content decreases with doubling time. Carbon-rich storage materials, polysaccharides and lipids, are more abundant at low growth rates, particularly under nitrogen limitation.
13 Data on individual macromolecules from different filamentous fungi appear contradictory (e.g. Griffen, D.H. et al, J. gen Microbiol. (1974), 80, 381; Bu'Lock, J.D. et aI, Trans. Br. mycol, Soc. (1974), 62,377). When such results are considered in the context of specific cellular functions, however, some uniformity in the effects of growth rate on hyphal macromolecular content becomes apparent. An expression for ribosomal efficiency has been proposed (Alroy, Y. and Tannenbaum, S.R., Biotech, Bioeng. (1973), 15,239) involving the ratios of protein, RNA and DNA productivity in terms of the relative specific growth rate. Subsequent calculations by the author have revealed linear relationships between ribosomal efficiency and growth rate per se, The appropriate plots passed through the origin in prokaryotes and cut the abscissa at the minimum growth rate for purely vegetative growth in fungal cultures capable of differentiation. This observation therefore, supports the concept of a minimum specific growth rate (Pirt, S.J., Appl. Chem. Biotech. (1972),22, 55). Studies with different species on the variation of hyphal lipid and carbohydrate content also appear contradictory when considered in isolation. In Aspergillus nidulans no simple relationship between intracellular concentration and doubling time was apparent for either polymer. When the combined energy storage equivalent value (Forrest, W.W. and Walker, DJ. (Adv. Microb. Phys. (I971), 5, 213) was calculated however, a linear relationship between energy value and growth rate was established. Growth related changes in ribosomal efficiency and DNA productivity may be related to corresponding alterations in hyphal polyamime content. Attempts to deduce ad hoc relationships between polyamine content and fungal morphogenises have been unsuccessful (Marshall, M. et al, Curro Microbiol. (I979) 2, 187). Evidence for a more fundamental connection between polyamines and fungal growth will be presented. Factors affecting the growth of dimorphic fungi. F.W. Chattaway , Department of Biochemistry, University of Leeds, Leeds, LS2 9LS. The control of morphological development in dimorphic fungi is of intrinsic interest in relation to the role of environmental factors and of probable interest in the pathogenicity of some species in mammalian hosts. The cell wall may have a role in determining morphology and differences have been shown in the composition of cell wall from yeast and mycelial forms in terms of glucan, chitin and protein content. Environmental factors which can affect morphological development are temperature, pH, oxygen supply, glucose concentration and the nature of the nitrogen source in the medium; in addition the size of the inoculum and the age of cells used can be modifying factors. Biochemical systems which have been studied as possibly affecting the observed type of growth are (i) chitin synthetase in Candida albicans, (ii) glycolysis and related pathways in Mucor spp. and Candida albicans, (iii) protein synthesis in relation to sulphur metabolism and RNA polymerases in Histoplasma capsula tum . Biochemical activity in many organisms is regulated by cyclic -3', 5' -adenosinemonophosphate, and there is increasing evidence for its possible role in controlling dimorphism. The autolytic phase of growth in filamentous fungi: shape and size of vacuoles of Neurospora crassa. R. Lehoz and F. Reyes, Instituto 'Jaime Ferran' de Microbiologia del e.S.I.C., Joaquin Costa, 32, Madrid-6, Spain. Changes in the shape and size of vacuoles of Neurospora crassa, as seen under the light microscope, in relation to autolysis have been studied. Of a total hyphal length amounting to 22,600 11m 2,970 vacuoles were drawn, counted and
14 measured during pre-autolysis and autolysis, being 66.3% apparently circular or very nearly circular shaped. In the period between the first 24-36 hours incubation the mean vacuolar radius underwent an increase in length amounting to 44.4%. As incubation proceeded a lateral elongation of most of these circular vacuoles seems to take place. At the end of the log phase the ratio circular/non-circular vacuoles amounted to 8.1: 1; during the pre-autolytic stages this ratio decreased to 2.4: 1, being 0.4: 1 at the end of autolysis. Plotting the values of this ratio against time of incubation it yielded a hyperbolic curve. Measuring vacuolation as the change in the area of vacuoles, it has been observed that from a total hyphal area of 131,534 IJ.m 2 (measured in 369 hyphae) the vacuolar area increased nearly 25% during the pre-autolytic period, and 30% during autolysis, therefore a continuous increase of this vacuolar area with time of incubation occurred.
Control of growth and development by volatile metabolites. P.M. Robinson, Department of Botany, The Queen's University, Belfast, BT7 1NN. All stages of development of Geotrichum candidum can be influenced by selfproduced volatile metabolites. Arthrospores fail to germinate when seeded at a concentration of ~ lOs arthrospores ml- 1 in distilled water or distilledwater agar and this self-inhibition of germination is due to the production of volatile metabolites which retard germination when present at low concentrations. The increase in concentration of these metabolites which occurs with increasing arthrospore concentration parallels the increase in the level of selfinhibition of the arthrospore population and the associated decrease in negative autotropism of arthrospore pairs. The autotropic response may be due to or influenced by the volatile metabolite(s) responsible for self-inhibition. Hyphal development in G. candidum is affected by volatile metabolites produced by germinated or ungerminated arthrospores. Arthrospores exposed to these metabolites produce hyphae which sporulate earlier than untreated hyphae. At high concentrations of these metabolites hyphal extension is arrested abruptly and arthrospore formation does not occur; a decrease in the concentration of these metabolites results in continued extension-growth of the hyphae. The metabolites responsible for the affects described have not been characterized but preliminary work has established that the effects are not due to oxygen deficiency or to the production (or absence) of carbon dioxide.
Growth rate and metabolic control in yeast. D.R. Berry, Department of Applied Microbiology, University of Strathclyde, George Street, Glasgow, GI lXW. It is well established that the levels of carbon, nitrogen and phosphorus in the growth medium markedly influence the growth and development of microorganisms. In yeast the role of glucose in catabolite repression is well established and nitrogen depletion has been reported to influence glycogen storage. The levels of carbon, nitrogen and phosphorus are also known to be important in controlling the quality of bakers yeast. In this paper the effect of variations in the level of glucose, ammonium and phosphate on the metabolism of yeast have been studied. Techniques have been developed for the rapid and accurate analysis of intermediates of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and the glyoxylate cycle in yeast grown in batch and continuous culture. The application of the techniques to batch culture gave complex results.
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The use of continuous culture provided stable , precisely defined physiological conditions for studying the level of intermediates. The results indicated that intermediate levels in the pathways studied are a function of the nutritional status of the medium and of the growth rate. They appeared to be stable for a given steady state , however fluctuations in the levels of intermediates could be induced by the add ition of nutrients in short pulses .
Carbohydrate metabolism and morphogenesis. D. Moore, Department of Botany, University of Manchester, Manchester, Ml3 9PL. The varied relationship between carbohydrate metabolism and fungal morphogenesis will be illustrated by discussion of a number of different examples. The part played by enzymes responsible for interconversion of sugars is clearly important , since mutations in genes specifying these enzymes often lead to morphological abnormalities of hyphal and vegetative colony growth. Similar abnormalities can also be induced by inhibitors and experiments with the latter represent a means of studying the way in which hyphal form can be modified. During normal morphogenesis changes which represent steps in development are precipitated by both environmental and metabolic events. Agents which might be involved in signalling such changes include the ubiquitous cyclicAMP, but may also include metabolic intermediates. This point will be illustrated by discuss ion of the possible role of pyruvate in the regulation of glutamate dehydrogenase during vegetative growth of a strain of Coprinus cinereus in relation to events which occur endogenously during morphogenesis of the carpophore cap.
Genetic approaches to the control of nitrogen metabolism in Aspergillus nidulans . H.N . Arst , Jr ., Department of Genetics, University of Newcastle upon Tyne , Newcastle upon Tyne, NEI 7RU . The syntheses of many enzymes and permeases which playa role in the nitrogen nutrition of Aspergillus nidulans are subject to one or more forms of regulation . These include induction, nitrogen metabolite (i.e. ammonium) repression, and, where there is an involvement in carbon nutrition, carbon catabolite repression. All of the regulatory genes mediating induction investigated to date are positive acting and can be classified as pathway-specific or integrator (i.e . mediating concomitant induction of activities coded by two or more non-contiguous structural genes of which at least one can also be expressed independently by an alternative induction mechanism). Nitrogen metabolite repression is mediated by a positive acting regulatory gene designated areA , and ammonium (or a metabolite derived from it) inactivates (or less likely represses formation of) the are A product. In at least some cases carbon catabolite repression is mediated by a negatively acting regulatory gene designated creA. Cis-acting receptor sites for these regulatory gene products have been identified by mutations in a few cases. In one case a new regulatory pattern has arisen from fusion of a structural gene to a promoter normally located in a different linkage group as the result of an insertional translocation. As in other eukaryotes , functionally related genes are seldom clustered in A . nidulans. Some exceptions to thi s generalisation include 'cluster-genes' coding for polyfunctional polypeptides and a few bona fide gene clusters whose significance and genetic organisation is under study. In one such cluster, there is genetic evidence for synthesis of a dicistronic transcript plus at least one (mono-, tri- , or tetracistronic) overlapping transcript .
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Biosynthesis of amino acids in yeasts and its exclusion from degradation . J-M. Wiame, Laboratoire de Microbiologie, Universite Libre de Bruxelles, et Institut de Recherches du CERIA, 1 avo E. Gryson, B-1070 Brussels, Belgium . Yeasts have the simultaneous possibility of synthesis and degradation of aminoacids. Occasionally some intermediary metabolites are common to both processes and bring up the problem of futile cycle exclusion . Experimental production of such a cycle by mutation has allowed us to appreciate the factors which avoid the occurrence of this waste in normal cells . In asparagine synthesis the exclusion is remarkably simple and stands on an adequate adjustment of enzyme kinetic parameters. The comparative study of a number of yeast genera illustrates the possibility of appreciating the physiological factors which may have been involved in the selection of two types of futile cycle avoidance. In arginine metabolism , the occurrence of an epiarginasic regulation versus a pro cess of compartmentation involving the mitochondrion appears to be correlative with energy metabolism . In addition to enzymes as the direct target, exclusion at the level of an enzyme synthesis is also illustrated in arginine metabolism at the level of genetic regulatory circuits. In that case a repressor ARGR has two distinct functions (ambivalent repressor): it represses as usually the biosynthetic enzymes in binding with operators and, in addition, it removes the catabolic specific repressor CARG R from the operators of the two genes coding for catabolic enzymes. The dual function of ARGR is nicely shown by a unique mutation which favours catabolism (and is dominant over wild type) and simultaneously weakens its anabolic role and is recessive.
Control of chitin synthesis. G.W. Gooday , Department of Microbiology , Marischal College, University of Aberdeen, Aberdeen, AB9 1AS. The activ ity of the enzyme chitin synthase is affected by a range of metabolites and cell constituents. These include uridine-containing molecules , especially the substrate UDP-N-acetylglucosamine and the product uridine diphosphate; the monomer and dimer of chitin , N-acetylglucosamine and N ,N '-
Control of lipid metabolism in y east and fungal growth . Anthony H. Rose, Zymology Laboratory, School of Biological Sciences, University of Bath, Bath, Avon . Yeast and fungal lipids are made up of two classes. The polar lipids include phospholipids and sterols, which are located in the plasma membrane with intracellular membranes. The non-polar lipids, which include triacylglycerols and sterol esters, are located not in membranes but in intracellular low-density vesicles . Overproduction of phospholipids and sterols is alm ost ne ver observed, but many yeasts and fungi are able to produce large amounts of neutral lipids, so metimes accounting for as much as 60% of the dry weight of the biomass. Metabolic pathways have been charted for synthesis of all of the major classes of yeast and fungal lipids , and there is evidence that the amounts synthesized are regulated by well established feedback processes. However , many problems remain unsolved in the biosynthesis of yeast and fungal lipids . The two major phospholipids , namely phosphatidylcholine and phosphatidylethanolamine, can
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be synthesized by either the cytidine nucleotide or the methylation pathway. Little is known of the metabolic factors which dictate which of the pathways is used for each of these phospholipids. Nor is there more than a cursory understanding of the mechanisms which regulate the fatty-acyl composition of phospholipids , triacylglycerols and sterol esters. While the subcellular location of the various classes of lipid has been established , that of the sites in the cell where the various lipids are synthesized is unclear. Finally , while there is evidence for a role for neutral lipids in biosynthesis of polar lipids , information on this role is meagre .
Some relationships between primary and secondary metabolism . M.O. Moss, Department of Microbiology, University of Surrey, Guildford, Surrey, GU2 5XH. Several decades of intensive study of the chemistry of secondary metabolites have uncovered a considerable diversity of structures. At the biochemical level, it has been possible to see patterns in this diversity through the elucidation of biosynthetic pathways and the demonstration that relatively few fundamental pathways are involved in the production of thes e metabolites. It is at the biosynthetic level that the links between primary and secondary metabolites are most apparent for the biosynthesis of each involves the same relatively few low molecular weight precursor molecules . Thus acetyl coenzyme A is a precursor of both fatty acids and polyketides , amino acids are precursors of both proteins and diketopiperazines, [3-lactams and alkaloids, whereas mevalonate is a pre cursor of terpenes, sesquiterpenes and diterpenes as well as sterols and carotenoids. At the physiological level, the relationship between primary and secondary metabolism as time dependent processes is less certain. Any general theory of secondary metabolism must take account of the very large number of chemically distinct, and apparently genetically defined, compounds described as secondary metabolites as well as the specificity with which they are frequently associated with individual species of microorganism. It must also take account of the physiological state of the producing organism at the time of optimum pro duction of secondary metabolites , as well as the often whimsical nature of the production of particular compounds by some species. The pharmaceutical industry has adequately demonstrated that it is possible to manipulate both the genetic and physiological status of a species to optimise , and often very dramati cally increase, the yield of a particular metabolite with useful pharmaceutical properties . Bearing these points in mind, this paper will consider some speculative aspects of the natural history of primary and secondary metabolism.
Genetic control of penicillin produ ction. K.D. Macdonald , Chemical Defence Establishment , Porton Down , Salisbury , Wilts, SP4 OJQ . Penicillin is elaborated by a number of fungi including Aspergillus nidulans and Penicillium chrysogenum . The latter is employed for the industrial production of penicillin and the possibility of using the parasexual mechanism it possesses to breed improved strains has been explored. Aspergillus nidulans , which has both sexual and parasexual cycles , yields much less penicillin than Penicillium chryso genu m but because its formal genetics is much better understood it is a useful model organism for genetic studies on penicillin production. An examination of mutants of Penicillium chrysogenum impaired in penicillin biosynt hesis established that there were five different lo ci involved and a similar number of loci were found in Aspergillus nidulans. Studies with mutants of Penicillium chrysogenum which overproduced penicillin indicated that these were usually recessive. When attempts were made
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to cross mutants from divergent lines it was found that most of the progeny had one or other parental genome probably due to different chromosomal rearrangements in the parental strains. The lack of recombination and thus an inability to isolate progeny with better penicillin yields than either parent was overcome by avoiding mutagens causing gross chromosomal damage and crossing strains not widely divergent in lineage. Three mutants of Aspergillus nidulans with increased penicillin yields were positioned on three different linkage groups . Crosses indicated that one was recessive , one semi-dominant and the third , dominant. The recessive mutant was epistatic to the other two , and the semi-dominant mutant epistatic to the dominant mutant. Crosses between different wild type strains of As pergillus nidulans yielded progeny with better penicillin yields than either parent.
Physiological aspects of my cotoxin production in f ermenter conditions. I.E. Smith , Department of Applied Microbiology, University of Strathclyde, George Street, Glasgow, G I I XW. Aflatoxins are a group of related difunaro-coumarin secondary metabolites which are produced by Aspergillus [lavus and Aspergillus parasitic us. These mycotoxins are acutely toxic and have been shown to be carcinogenic to certain anim al species. Aflatoxins normally enter the dietary chain by way of agricultural products contaminated by toxin producing moulds. Much interest has been con cerned with the effect of various acids , namely propionic acid , on controlling the growth of these organisms and toxin production . The present paper will attempt to study some aspects of aflatoxin production by Aspergillus flavus in liquid and solid state fermentation conditions. In particular, these results will show propionic acid at certain concentrations promotes a stimulation of aflatoxin production by the producer organism. These effe cts have been studied in liquid and in solid surface cultivation. In particular, these studies show that the stimulated effect of propionic acid occurs during the early hours of spore germination. Propionic acid has no effect on an actively growing vegetative culture.
Control of ergot alkaloid biosynthesis. P.G . Mantle , Department of Biochemistry , Imperial College , London , SW7 2AY. Ergot fungi almost invariably produce their characteristic alkaloids while growing as plant parasites but the yields are generally less than 0.5%. In some species clavine alkaloids are produced apparently wit hout any particular differentiation of a special growth form . Alkaloid production in submerged cultur e is therefore fairly readily achieved provided that glucan accumulation is controlled . In other species, noted for their ability to elaborate the more complex alkaloids (the lysergic acid derivatives), the parasitic sclerotial tissue is quite different from any other tissue formed by the fungus. In Clav iceps purpurea , for example, sclerotial cells contain a triglyceride oil which is not produced by any other micro-organism and alkaloid is only biosynthesised by these lipid-rich cells . The control of sclerotial-like growth in axenic culture is a prerequisite for alkaloid production. Certain isolates of C. paspali have been manipulated in submerged culture to over-produce a lysergic acid isomer, or simple derivatives, by a factor of about 10" but this may reflect suppression of pathways normally diverting tryptophan into other secondary metabolites . Alkaloid production by C. purpurea in axenic culture is rare and the direct fermentation production of therapeutically-valuable alkaloids by this organism presents the principal challenge concerning understanding and applying control of alkaloid biosyntheses.
19 The main pathways of peptide alkaloid biosynthesis will be discussed and the extent to which enzyme systems will accept foreign precursor analogues will be illustrated by describing the bioproduction of a new cyclic tripeptide alkaloid analogue.