A Phytophthora infestans G-protein beta subunit is involved in sporangium formation

The heterotrimeric G-protein pathway regulates cellular responses to a wide range of extracellular signals in virtually all eukaryotes. It also controls various developmental processes in the oomycete plant pathogen Phytophthora infestans, as was concluded from previous studies on the role of the G-protein α-subunit PiGPA1 in this organism. The expression of the P. infestans G-protein β-subunit gene Pigpb1 was induced in nutrient-starved mycelium before the onset of sporangium formation. The gene was hardly expressed in mycelium incubated in rich growth medium. The introduction of additional copies of Pigpb1 into the genome led to silencing of the gene and resulted in transformants deficient in PiGPB1. These Pigpb1-silenced mutants formed very few asexual spores (sporangia) when cultured in rye sucrose medium and produced a denser mat of aerial mycelium than the wild type. Partially Pigpb1-silenced mutants showed intermediate phenotypes with regard to sporulation, and a relatively large number of their sporangia were malformed. The results show that PiGPB1 is important for vegetative growth and sporulation and, therefore, for the pathogenicity of this organism.     

Synthesis of different pectinases by filamentous growingA. niger mutants

Mutants ofA. niger K 69/26, prepared by multistep mutagenesis (UV, MNNG, heating) have been screened for pectinase activities. Mutants with altered levels of certain pectinases, such as endo- and exopolygalacturonase (PG vis, red), pectinesterase (PE) and pectinlyase (PL), were isolated. The enzyme activities of the best mutants M 1348/126 were increased 2–3-fold compared to the parent strain after a 6-d cultivation of filamentous mycelium on a shaker. Further mutagenesis of mutants with decreased pectinase activities (e.g. Se3) produced revertants. PG (vis) synthesis of revertant Se5 was increased 1.7 times compared to the control strain K 69/26. Independent of these increased rates, the general level of pectinase activities synthesized by the filamentous mycelium ofA. niger mutants amounts to about 10–20% compared with those produced by aggregated mycelium. It appears that the enzyme synthesis related to mycelium structure is independent of the mechanism which regulates the level of pectinase synthesis within a specific morphological structure.     

Replisome localization in vegetative and aerial hyphae of Streptomyces coelicolor

Using a functional fusion of DnaN to enhanced green fluorescent protein, we examined the subcellular localization of the replisome machinery in the vegetative mycelium and aerial mycelium of the multinucleoid organism Streptomyces coelicolor. Chromosome replication took place in many compartments of both types of hypha, with the apical compartments of the aerial mycelium exhibiting the highest replication activity. Within a single compartment, the number of "current" ongoing DNA replications was lower than the expected chromosome number, and the appearance of fluorescent foci was often heterogeneous, indicating that this process is asynchronous within compartments and that only selected chromosomes undergo replication.     

Lectins in higher fungi

The occurrence of lectins in higher fungi, localisation in the sporome and mycelium, levels according to different parameters and roles in the organism are reviewed. Chemical structure and specificity of lectins are examined. Applications of fungal lectins are discussed.     

Selenocystine-resistant mutants of Histoplasma capsulatum

Cysteine metabolism has been thought to be important to the phenomenon of dimorphism inHistoplasma capsulatum. We sought mutants with genetic blocks in the metabolism of cysteine by selection of colonies resistant to the toxic analogue, selenocystine. The 22 resistant strains thus obtained were all deficient in uptake of cystine from the surrounding medium but were normally able to convert from mycelium to yeast and back again. Furthermore, they had normal quantities of NADH-dependent cystine reductase when this enzyme was measured. We conclude that mutants defective in cystine uptake can be readily obtained by selection of colonies resistant to selenocystine, and that a lesion in cystine-uptake does not appear to affect the phenomenon of dimorphism in this organism.Preliminary reports of this work were presented at the Second International Congress of Mycology, Tampa, 1977 and at the first International Conference on Histoplasmosis, Atlanta, 1978.     

Isolation and properties of mutants of the fungus Penicillium pinophilum with enhanced cellulase and β-glucosidase production

A number of mutant strains overproducing cellulase, β-glucosidase and xylanase enzyme were isolated from the cellulolytic fungus Penicillium pinophilum 87160iii after mutagenesis by u.v. irradiation and/or chemical treatment. Selection was carried out using either an agar-plate or an enrichment technique. Cellulase (filter paper-hydrolysing activity) production by some of the mutants in shake flask cultures was approximately four-fold higher than the wild-type strain; improvements in β-glucosidase production were of the order of eight- to-ninefold. The morphology of the mycelium of the mutants was quite different from that of the wild type. The mutants, for example, produced mycelium which was highly branched and thicker in cross section. In several of the mutants synthesis of xylanase and β-glucosidase was completely derepressed in the presence of glycerol, which was a known repressor of the synthesis of these enzymes. Several of the mutants produced β-glucosidase enzyme which showed altered kinetics of hydrolysis in the presence of inhibitors.     

Release of proteinase from mycelium of Mucor hiemalis

When Mucor hiemalis NRRL 3103 was grown in soybean medium, only a small fraction of the proteinase produced by the organism appeared in the culture filtrate, whereas the bulk of the enzyme was bound to the mycelial surface. Optimal pH of the proteinase ranged from 3.0 to 3.5. Inclusion of sodium chloride or other ionizable salts in the growth medium, however, resulted in the liberation from the mycelium of the loosely bound enzyme as it was formed. Maximal release of proteinase was achieved at a sodium chloride concentration of 0.5 m. The loosely bound proteinase was eluted also from intact resting mycelium by ionizable salts but not by water or by nonionizable substances. The amount of enzyme eluted from the mycelium depended upon the concentration of sodium chloride up to 0.3 m. Since liberation took place rapidly even at 0 C, a loose ionic linkage must exist rather than a biochemical binding of the enzyme to the mycelium. The recovery of proteolytic activity from repeated salt extractions was greater than that originally detected in the intact mycelium, possibly owing to unmasking of more active enzymes or functional groups. Further proteinase activity was released when salt-extracted mycelium was ruptured. Part of the proteinase thus observed was firmly attached to the cell fraction, and part of it appeared in the supernatant fluid. These conditions implied the presence of intracellular or firmly attached proteinase which could be partially released.     

Morphology transition genes in the dimorphic fission yeast <Emphasis Type="Italic">Schizosaccharomyces</Emphasis><Emphasis Type="Italic">japonicus</Emphasis>

The ability of the saprophytic fungus Schizosaccharomyces japonicus to alternate between unicellular yeast form and multicellular true mycelium makes this organism an attractive non-pathogenic model for the investigation of di- and polymorphism critical for pathogenicity in many pathogenic fungi. In a previous work we described three mutations that made the cells unable to form hyphae. Here we report on the isolation of additional mycelium-minus mutants and show that the mutations represent seven genes. Apart from the inhibition of the yeast-to-mycelium transition, the mutations also cause drastic changes in the yeast phase. Cells of myc3-34 and myc4-35 grow isotropically with apolar distribution of actin and randomised localisation of division planes. The mutants myc1-4, myc1-10, myc1-36, myc5-39 and myc6-43 form sep-like, branching microhyphae containing bipolarly growing cells. All mutants are defective in the polarisation of vacuole distribution, and myc3-34, myc4-35 and myc7-56 are also defective in vacuole fusion. The diversity of the mutant phenotypes indicates that several cellular processes must take place simultaneously for the transition from the yeast phase into the mycelial phase.     

Study of the fructosediphosphate aldolase and transketolase activity in the nystatin producer, Actinomyces noursei]

Activity of transketolase, an enzyme of the pentose cycle and fructosodiphosphataldolase, an enzyme of glycolisis was studied in the dynamics of development of the nystatin-producing organism and its inactive mutant under various conditions of their cultivation with a purpose of finding relation between the antibiotic production and general metabolism of Act. noursei. The transketolase activity of the organism was 2-4 times higher than that of the inactive mutant. Addition of 8000 Units/ml of nystatin to the medium markedly suppressed (50-100 per cent) the aldolase activity, however it had no effect on the transkelotase activity. Possibly the antibiotic accumulated in the mycelium played the role of a regulator of the activity of the enzymes, directing the metabolites along the hexosomonophosphate pathway of carbohydrate dissimilation.     

Stereospecific Metabolism of O-Ethyl O-2-Nitro-5-methylphenyl N-Isopropyl Phosphoramidothioate (S-2571) by Liver Microsomal Mixed Function Oxidase

The distribution of lipoprotein lipase activity in microorganisms was examined, Rhizopus japonicus KY 521 showed the highest activity of lipoprotein lipase in the culture fluid among microorganisms tested, Lipase was also excreted in addition to lipoprotein lipase by this organism. The effect of cultural conditions on the extracellular production of the two lipases by this organism was investigated. The addition of phospholipid such as lecithin brought about a remarkable increase in the extracellular production of both lipases. It was found that lecithin did not increase significantly the net synthesis of the enzyme, but accelerated the secretion of the enzyme formed in the mycelium into the culture medium.     

Ultrastructure of the mycelium and spores of Actinomadura fastidiosa sp. nov]

A new species of the Actinomadura genus, A. fastidiosa sp. nov., is described. The ultrastructure of the vegetative mycelium and spores of this organism was studied. The vegetative cells have a multilayered cell wall, often consisting of five layers with different thickness and electron density. The spores are similar to the vegetative cells by their inner structure but have a thicker wall.     

Cellulolytic Ability of the Scab Fungus, Venturia inaequalis

Cellulase and β-D-glucosidase activity have been identified in Venturia inaequalis, the causal agent of apple scab. Most of the activity of the enzymes was associated with the mycelium after homogenization. The activities were relatively weak in vitro compared to those of other plant pathogens. With a new screening technique, β-D-glucosidase hyperproducing mutants were obtained.     

Changes in patterns of ADP-ribosylated proteins during differentiation of Streptomyces coelicolor A3(2) and its development mutants.

Mutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces coelicolor A3(2). One (strain 27) was analyzed in detail. Mutant 27 had a reduced ADP-ribosyl-transferase activity, exhibited substantial changes from the wild type in ADP-ribosylated protein profile during cell aging, and was defective in producing aerial mycelium and antibiotics. A 92-kDa ADP-ribosylated protein disappeared at the onset of differentiation in the parent strain but was present in mutant 27. Four ADP-ribosylated proteins (39, 41, 43, and 46 kDa) appeared at the onset of differentiation in the parent strain but were missing in mutant 27. Failure to ADP-ribosylate these four proteins was detected when the parent strain was grown in the presence of subinhibitory amounts of 3-aminobenzamide. Genetic analysis showed that the mutation, named brgA, conferring resistance to 3-aminobenzamide, cosegregated with the altered phenotypes (i.e., defects in ADP-ribosylation and aerial mycelium formation) and was mapped to a new locus near uraA. The brgA mutants were nonconditionally deficient in producing aerial mycelium and antibiotics, as determined by using various media, and had a morphological and physiological phenotype quite different from that of a bldG mutant carrying a mutation which was previously mapped near uraA. Among the known bld mutants, bldA, bldD, and bldG mutants exhibited a ADP-ribosylated protein profile similar to that of the wild type, while like mutant 27, bldB, bldC, and bldH mutants failed to ADP-ribosylate certain proteins.     

Study of the chemical makeup of the mycelium from an active strain of Act. rimosus and from an inactive mutant in relaiton to oxytetracycline biosynthesis]

Chemical composition of the mycelium of the active and inactive mutants of Act. rimosus grown under conditions favourable for oxytetracycline biosynthesis on the starch or maltose medium and under favourable conditions on the glucose medium was studied. It was shown that according to its chemical composition the above strains did not practically differ. When grown on the starch medium the mycelium of both strains contained great amounts of carbohydrates and comparatively small amounts of nucleic acids and nitrogen. Replacement of starch in the medium by glucose or maltose induced significant changes in the mycelium composition: the synthesis of intracellular polysaccharides was markedly suppressed and the synthesis of nucleic acids and nitrogen containing compounds increased. RNA was the main nucleic acid in both strains on starch and glucose media. The content of DNA was low and did not practically change. The mycelium of both strains contained small amounts of lipids which did not significantly change during the process of cultivation and did not correlate with the antibiotic activity.     

Phosphoenolpyruvate:carbohydrate phosphotransferase systems in Enterococcus faecalis

A wild-type strain of Enterococcus faecalis and its mutants resistant to 2-deoxy-D-glucose (2DG) were examined for the presence of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs) with 12 carbohydrates, which were utilized by the organism, as the substrates. The wild-type strain possessed a constitutive mannose-PTS, which was reactive with glucose, mannose, glucosamine, 2DG and fructose. This activity was absent in the mutants. No independent glucose- or fructose-PTS was found in the mannose-PTS-defective mutants. The mutants, however, showed a low level of a constitutive PTS activity with maltose, suggesting the existence of an independent maltose-PTS in the organism. Both wild-type and mutant strains possessed inducible lactose-, mannitol-, and trehalose-PTSs. Lactose-PTS was induced by either lactose or galactose in the parent, but only by lactose in the mutants. The lactose-PTS was not reactive with galactose, and no separate galactose-PTS was present. These observations suggest that the inducer for lactose-PTS, probably being galactose 6-phosphate, may not be formed from galactose in the organism when the constitutive mannose-PTS is lost by mutation.     

A new genus of the Actinoplanaceae: Planobispora,gen. nov.

Summary A new species of a new genus of the Actinoplanaceae is described, for which the name Planobispora longispora gen. nov. sp. nov. is proposed. The organism is a typical mesophilic, aerobic actinomycete, producing a filamentous growth which is differentiated into a vegetative and an aerial mycelium. The new organism is characterized by the formation of sporangia only on the aerial mycelium and by containing a longitudinal pair of motile spores.     

Polymorphism of a culture of Actinomyces chromogenes var. trienicus, the producer of chromotrienine]

Variation of Actinomyces chromogenes var. trienicus 141-18 MSU, an organism producing trienin was studied under laboratory conditions. Nine stable spontaneous variants were isolated from the population of the initial culture when grown on Gause medium No. 1. The variants varied in differentiation and biosynthetic capacity, including such characteristics as size and form of the colonies, ability for formation of the aerial mycelium and its colour, capacity for sporulation, form of the spore chains and antibiotic production property. In the secondary structures the spores formed only in 6 variants out of 9 isolates. The spore form and spore membrane surface were close in all sporogenic variants, while there were significant differences in the structure of the sporophores. The variants forming the aerial mycelium of the same colour as that of the initial culture did not differ from it also by the nature of the spore chains (spirals with 3--8 turns). The variants with lighter aerial mycelium than that of the initial population formed straight sporophores or spirals with a small number of the turns (1--3). The comparative study of the antimicrobial spectrum of the variants and the component composition of the synthesized antibiotic complex showed that the asporogenic variants and dwarf variant signifcantly differed with respect to their phenotypes from the other cultures and had no antagonistic action. One of the assporogenic variants had only insignificant activity. All the spore forming variants did not differ from the initial culture in the complex of the antibiotics synthesized.     

Regulation of biosynthesis of secondary metabolites

The biosynthetic activity of mutants with altered morphological and biochemical characteristics, obtained from two strains ofStreptomyces aureofaciens by means of physical and chemical mutagens, was studied. The majority of mutants formed pigments different from pigments of the parent strains, which did not usually give fluorescence in UV-light and, in addition, differed as to the solubility in organic solvents. The production of further secondary metabolites was investigated chromatographically in the extracts from mycelium and in the fermentation fluid of submerged cultures. According to the results of chromatographical analysis, the obtained mutants can be divided into 12 metabolic types. In most of them the production of substances was found which are different both mutually and from the metabolites of parent strains in physical and chemical properties. A direct correlation was observed between the character of colonies and biosynthetic properties of mutants.     

Study of the carbohydrate content in the mycelia of an active strain producing oxytetracycline and in an inactive mutant]

The content of carbohydrates in the mycelium of the active strain and inactive mutant of the oxytetracycline-producing organism under conditions favourable (starch medium) and unfavourable (glucose medium) for the antibiotic biosynthesis was studied. The mycelium of both organisms was fractionated and carbohydrate distribution according to the mycelium fractions and carbohydrate content in every fraction were investigated. No significant differences were observed between the active strain and inactive mutant with respect to the characteristics studied. The carbon source in the medium had the dominating effect on the chemical composition of the mycelium. The mycelium of both strains grown on the starch medium contained much more carbohydrates than that grown on the glucose medium. The carbohydrates of the mycelium grown on the starch medium were mainly found in fraction III and must be represented by polysaccharides.     

Metabolic Role, Regulation of Synthesis, Cellular Localization, and Genetic Control of the Glyoxylate Cycle Enzymes in Neurospora crassa

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora, isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.