Biomedical subjects

G D Robson

Publications and source records attributed to G D Robson.

35 records · Page 2Linked to original sources

PubMed1999

pH regulation of recombinant glucoamylase production in Fusarium venenatum JeRS 325, a transformant with a Fusarium oxysporum alkaline (trypsin-like) protease promoter.

Fusarium venenatum (formerly Fusarium graminearum) JeRS 325 produces heterologous glucoamylase (GAM) under the regulation of a Fusarium oxysporum alkaline (trypsin-like) protease promoter. The glucoamylase gene was used as a reporter gene to study the effects of ammonium and pH on GAM production under the control of the alkaline protease promoter. Between pH 4.0 and 5.8, GAM production in glucose-limited chemostat cultures of JeRS 325 grown at a dilution rate of 0.10 h-1 (doubling time, 6.9 h) on (NH4)2SO4 medium increased in a linear manner with increase in pH. However, at pH 4.0 and below GAM production was almost completely repressed in glucose-limited chemostat cultures grown on (NH4)2SO4 or NaNO3 medium. Thus GAM production in JeRS 325 is regulated by culture pH, not by the nature of the nitrogen source in the medium. The difficulty of using unbuffered medium when investigating putative ammonium repression is also shown. The study demonstrates the potential for use of the alkaline protease promoter in F. graminearum for the production of recombinant proteins in a pH dependent man ner.

Cells, Cultured↗

PubMed1999

Plasticizers increase adhesion of the deteriogenic fungus Aureobasidium pullulans to polyvinyl chloride.

Initial adhesion of fungi to plasticized polyvinyl chloride (pPVC) may determine subsequent colonization and biodeterioration processes. The deteriogenic fungus Aureobasidium pullulans was used to investigate the physicochemical nature of adhesion to both unplasticized PVC (uPVC) and pPVC containing the plasticizers dioctyl phthalate (DOP) and dioctyl adipate (DOA). A quantitative adhesion assay using image analysis identified fundamental differences in the mechanism of adhesion of A. pullulans blastospores to these substrata. Adhesion to pPVC was greater than that to uPVC by a maximum of 280% after a 4-h incubation with 10(8) blastospores ml(-1). That plasticizers enhance adhesion to PVC was confirmed by incorporating a dispersion of both DOA and DOP into the blastospore suspension. Adhesion to uPVC was increased by up to 308% in the presence of the dispersed plasticizers. Hydrophobic interactions were found to dominate adhesion to uPVC because (i) a strong positive correlation was observed between substratum hydrophobicity (measured by using a dynamic contact angle analyzer) and adhesion to a range of unplasticized polymers including uPVC, and (ii) neither the pH nor the electrolyte concentration of the suspension buffer, both of which influence electrostatic interactions, affected adhesion to uPVC. In contrast, adhesion to pPVC is principally controlled by electrostatic interactions. Enhanced adhesion to pPVC occurred despite a relative reduction of 13 degrees in the water contact angle of pPVC compared to that of uPVC. Furthermore, adhesion to pPVC was strongly dependent on both the pH and electrolyte concentration of the suspension medium, reaching maximum levels at pH 8 and with an electrolyte concentration of 10 mM NaCl. Plasticization with DOP and DOA therefore increases adhesion of A. pullulans blastospores to pPVC through an interaction mediated by electrostatic forces.

Ascomycota↗

PubMed1998

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture.

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.

Aspergillus niger↗

PubMed1998

Recombinant glucoamylase production by Aspergillus niger B1 in chemostat and pH auxostat cultures.

The production of glucoamylase (GAM) by Aspergillus niger B1, a genetic transformant containing an additional 20 copies of the homologous glucoamylase gene (glaA) was studied in nutrient (maltodextrin)-limited chemostat and nutrient-excess pH auxostat cultures. In these culture systems the specific production rate of GAM increased with dilution rate and reached a maximum (up to 15.0 mg GAM [g biomass]-1 h-1) when A. niger B1 was grown at its maximum specific growth rate in pH auxostat culture, indicating that GAM is a growth-associated product. The appearance of spontaneous morphological mutants was observed in all continuous flow cultures grown at pH 5.4, with a light brown mutant always displacing the parental strain. However, no morphological mutants were observed in cultures grown at pH 4.0. Further, when A. niger B1 was grown in pH auxostat culture, the specific production rate of GAM was 31% higher at pH 4.0 than at pH 5.4. Southern blot analyses showed that some morphological mutants (including the light brown mutant) isolated from a pH auxostat culture had lost copies of the glaA genes.

Aspergillus niger↗

PubMed1994

Use of a series of chemostat cultures to isolate 'improved' variants of the Quorn mycoprotein fungus, Fusarium graminearum A3/5.

Variants (designated A23-S and A24-S) of the Quorn myco-protein fungus, Fusarium graminearum A3/5 were isolated from a series of glucose-limited cultures grown at a dilution rate of 0.18 h-1 for a combined total of 109 d. These variants had unchanged mycelial morphologies but, when grown in mixed culture with the parental strain (A3/5) in glucose-limited chemostat culture at 0.18 h-1, A23-S and A24-S had selection coefficients of 0.013 and 0.017 h-1, respectively, and supplanted A3/5. When a monoculture of A23-S was grown in a glucose-limited culture at a dilution rate of 0.18 h-1, the appearance of highly branched (so-called colonial) mutants was delayed compared with their appearance in chemostat cultures of the parental strain. Furthermore, when a monoculture of A24-S was grown in glucose-limited culture at 0.18 h-1, the appearance of colonial mutants was delayed even further. Thus, it is possible to isolate advantageous (relative to A3/5) variants of F. graminearum A3/5 which have unchanged mycelial morphologies, but in which the appearance of colonial mutants is delayed.

Cycloheximide↗

PubMed1994

Evolution of Fusarium graminearum A3/5 grown in a glucose-limited chemostat culture at a slow dilution rate.

The evolution of Fusarium graminearum A3/5 grown in a glucose-limited chemostat at a dilution rate of 0.05 h-1 (doubling time of 13.9 h) was followed for 957 h or 69 generations. Periodic selection of advantageous mutants was monitored in the culture by determining increases and decreases in the concentration of cycloheximide-resistant macroconidia in the population. Six peaks in the concentration of cycloheximide-resistant macroconidia were observed representing five adaptive changes in the population; on average, an adaptive change occurred once every 148 +/- 22 h (mean +/- SE). The selection coefficient of strains present at the start of each increase in the concentration of cycloheximide-resistant macroconidia (i.e. after the establishment of a new advantageous strain) was determined relative to A3/5 and was found to increase progressively with time. When grown at a dilution rate of 0.05 h-1, the strain (A28-S) isolated from the last adaptive peak had a selection coefficient of 0.023 h-1 relative to A3/5, but A28-S lost its selective advantage when grown at a dilution rate of about 0.11 h-1 and was at a selective disadvantage when grown at a dilution rate higher than 0.11 h-1. The Km value (12 +/- 5 microM) for uptake of glucose by A28-S was significantly lower than that for A3/5. The spontaneous mutation rate from cycloheximide sensitivity to cycloheximide resistance was estimated to be 1.8 (+/- 0.2) x 10(-6) h-1 or 2.5 x 10(-5) generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗

PubMed1994

Hydrocortisone-enhanced growth of Aspergillus spp.: implications for pathogenesis.

Aspergillus fumigatus and Aspergillus flavus are the most common cause of invasive mould infections worldwide and carry a high mortality. Corticosteroid therapy and Cushing's disease are associated with an increase in invasive aspergillosis. Corticosteroids impair immune function in mammals and, specifically, the conidicidal activity of human macrophages, which was thought to be sufficient explanation for this increased risk. However, we have found a 30-40% increase in growth rate of A. fumigatus and A. flavus exposed to pharmacological doses of hydrocortisone (a human glucocorticoid), suggesting an alternative or additional mechanism for the association. No significant effect was observed with other human steroids such as testosterone, oestradiol or progesterone, though a smaller (21%) but significant growth rate increase was obtained with the fungal sterol ergosterol. The presence of a ligand/receptor system is therefore possible in pathogenic Aspergillus spp. Although corticosterone-binding proteins have been identified in some yeast species, a demonstrable physiological effect has been lacking. Interruption of the putative ligand/receptor interaction could have a major effect on the growth and pathogenicity of A. fumigatus, providing opportunities for the development of alternative therapeutic strategies to those currently available.

Aspergillosis↗

PubMed1993

Multiple isomers of phosphatidyl inositol monophosphate and inositol bis- and trisphosphates from filamentous fungi.

The range of inositol phosphates and inositol phospholipids present in three filamentous fungi, Neurospora crassa, Fusarium graminearum and Phanerochaete chrysosporium has been investigated by HPLC analysis. The profiles obtained demonstrate that two isomers of phosphatidyl inositol monophosphate are present, and that an apparent complexity in the number of isomers of inositol bis- and trisphosphates is found in filamentous fungi that has not been observed in animal or plant cells.

Chromatography, High Pressure Liquid↗

PubMed1993

Periodic selection in longterm continuous-flow cultures of the filamentous fungus Fusarium graminearum.

By monitoring increases and decreases in the proportion of cycloheximide-resistant macroconidia, periodic selection was observed in populations of the filamentous fungus Fusarium graminearum, grown in glucose-limited chemostat cultures. The results indicated that periodic selection of advantageous mutants of F. graminearum occurred at intervals of about 124 h at both high (D = 0.19 h-1, approximately 34 generations) and low (D = 0.06 h-1, approximately 11 generations) dilution rates. Several 'adaptive' peaks (each indicating the appearance of an advantageous mutation) were observed before morphological (highly branched) mutants appeared in the populations; these mutants have previously been observed to have a selective advantage over the parental strain. At intervals, macroconidia harvested from the chemostat were used to inoculate plates of non-antibiotic-containing agar medium, and it was possible to monitor periodic selection in the original chemostat culture using second generation macroconidia harvested from these cultures. The proportion of cycloheximide-, potassium chlorate-, and p-fluoro-DL-phenylalanine-resistant macroconidia in these second generation macroconidia changed in a pattern similar to that observed when monitoring the proportion of cycloheximide-resistant macroconidia in the first generation population harvested directly from the chemostat. The experiments demonstrated that populations of filamentous fungi are heterogeneous and that much of this heterogeneity may already be present at the end of batch growth, i.e., before the onset of continuous cultivation.

Cycloheximide↗

PubMed1993

Inhibition of phosphatidylcholine and chitin biosynthesis in Pyricularia oryzae, Botrytis fabae and Fusarium graminearum by edifenphos.

Colony growth of the fungi Pyricularia oryzae, Botrytis fabae and Fusarium graminearum was reduced by 50% (ED50) by edifenphos concentrations of 7, 25 and 190 microM respectively; the phosphatidylcholine (PC) content of biomass of P. oryzae, B. fabae, and F. graminearum harvested from fungicide-containing-cultures was reduced by 50% by 6, 95 and 350 microM-edifenphos respectively. By contrast, the activities of membrane-bound chitin synthase preparations isolated from the three fungi were approximately equally sensitive to edifenphos. A direct relationship was observed between PC contents of biomass grown in the presence of ediphenphos and in vivo rates of chitin synthesis (biomass incubated with [3H]GlcNAc in the absence of fungicide). Membrane-bound chitin synthase preparations from P. oryzae grown in medium containing 3 or 6 microM-edifenphos had, at the same fungicide concentration, a lower rate of in vivo chitin synthesis than preparations isolated from biomass grown in the absence of edifenphos. Membrane-bound chitin synthase preparations from P. oryzae grown in the presence and absence of 6 microM-edifenphos had the same Km values for the substrate (UDP-[14C]GlcNAc) but different Vmax values. The results suggest that chitin synthesis is inhibited directly by non-competitive inhibition of chitin synthase activity, and indirectly following inhibition of PC biosynthesis. P. oryzae is very sensitive to edifenphos because inhibition of PC biosynthesis occurs at very low fungicide concentrations, and therefore in this fungus inhibition of PC biosynthesis probably represents the primary mode of action of the fungicide.

Antifungal Agents↗

PubMed1993

Choline: its role in the growth of filamentous fungi and the regulation of mycelial morphology.

Choline is an essential metabolite for the growth of filamentous fungi. It occurs most notably as a component of the major membrane phospholipid, phosphatidyl choline (lecithin), and fulfills a major role in sulphate metabolism in the form of choline-o-sulphate in many species. Choline is usually synthesised endogenously, but exogenous choline can also be taken up, either to compensate for metabolic deficiencies in choline-requiring mutants such as those of Aspergillus nidulans and Neurospora crassa, or as a normal function by species such as Fusarium graminearum which do not require added choline for growth. F. graminearum has a highly specific constitutive uptake system for this purpose. Recent studies have begun to indicate that choline also plays an important role in hyphal and mycelial morphology. Over a wide range of concentrations, choline influences mycelial morphology, apparently by controlling branch initiation. At high concentrations of added choline, branching is inhibited but specific growth rate is unaffected, leading to the production of rapidly extending, sparsely branched mycelia. Reduction of choline concentration allows a progressive increase in branching. Additionally, in choline-requiring mutants which have a very reduced content of choline, multiple tip-formation and apical branching occurs. Just prior to cessation of growth in choline-starved cultures of A. nidulans choline-requiring mutants, hyphal morphology changes due to a brief phase of unpolarised growth to produce spherical swellings called balloons, at or near hyphal apices. The precise mechanism by which choline affects fungal morphology is not yet known, although in A. nidulans it appears to be at least partially due to the influence of membrane composition on the synthesis of the hyphal wall polymer chitin. Several hypotheses for the possible mode of action of choline in affecting fungal morphology are discussed here.

Cell Membrane↗

PubMed1992

Choline transport in Fusarium graminearum A 3/5.

Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM).

Biological Transport, Active↗

PubMed1991

Relationships between phosphatidylcholine content, chitin synthesis, growth, and morphology of Aspergillus nidulans choC.

The phosphatidylcholine (PC) content of Aspergillus nidulans choC was varied by growing the auxotroph in medium containing various concentrations of choline chloride. Direct linear correlations were observed between PC content and in vivo chitin synthase activity, between in vivo chitin synthase activity and mean hyphal extension rate, and between mean hyphal extension rate and hyphal growth unit length; hyphal growth unit length is a measure of hyphal branching. Further, there was a correlation between PC content and colony radial growth rate. Thus, membrane composition is an important determinant of both hyphal (and colony) extension rate and mycelial morphology.

Acetylglucosamine↗

PubMed1991

Exogenous cAMP and cGMP modulate branching in fusarium graminearum.

A study was made of the effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and choline on the morphology and growth of a wild-type strain (A 3/5) and a highly branched, 'colonial' mutant strain (C106) of Fusarium graminearum. Addition of up to 50 mM-cAMP or cGMP to the medium had no effect on the specific growth rate of strain A 3/5. For strain A 3/5, but not for strain C106, exogenous cAMP caused significant decreases in both mean hyphal extension rate (E) and hyphal growth unit length (G), i.e. cAMP caused mycelia of strain A 3/5 to branch profusely. By contrast, for both strains, cGMP caused significant increases in both E and G, i.e. exogenous cGMP caused mycelia to branch more sparsely. The effects of exogenous cGMP and choline in increasing E and G were synergistic, but the effects of cGMP and choline counteracted the effect of cAMP. The mutant phenotype of strain C106 was not correlated with altered levels of endogenous cAMP or cGMP.

Choline↗

PubMed1989

Effect of choline on the morphology, growth and phospholipid composition of Fusarium graminearum.

Studies were made of the growth kinetics, morphology and phospholipid composition of two strains of Fusarium graminearum, a wild-type strain (A3/5) and a highly branched variant (C106) which arose spontaneously during cultivation of A3/5. No significant difference was observed between the hyphal diameters of the two strains and therefore increased branching of C106 could not be explained in the terms of an increase in hyphal radius in the absence of a change in hyphal growth unit volume. The two strains had the same specific growth rate in batch culture and this was not affected by the addition of up to 1.5 mM-choline to the medium. However, choline increased the mean hyphal extension rate and colony radial growth rate of both strains and this response was correlated with the formation of mycelia which were more sparsely branched than mycelia grown on medium lacking choline. Addition of betaine, choline, ethanolamine, monomethylethanolamine or dimethylethanolamine (but not serine, glycine, dimethylglycine, methylamine, hydroxylamine or beta-hydroxyethylhydrazine) to the medium also resulted in appreciable increases in the colony radial growth rates of A3/5 (increased by about 130% for choline) and C106 (increased by about 25% for choline). No significant difference was observed between the phospholipid compositions of the two strains, and the addition of 100 microM-choline to the medium had no significant effect on the phospholipid composition of either strain.

Betaine↗

PubMed1988

Effects of validamycin A on the morphology, growth and sporulation of Rhizoctonia cerealis, Fusarium culmorum and other fungi.

All Basidiomycotina screened were sensitive to validamycin A, whereas most Ascomycotina and all Mucorales and Oomycetes were insensitive. Studies with Rhizoctonia cerealis and Fusarium culmorum showed that, in semi-solid culture, the antibiotic caused a decrease in colony radial growth rate and that this was associated with a decrease in mean hyphal extension rate and an increase in hyphal branching. However, the antibiotic did not alter the morphology of R. cerealis grown in liquid culture (shaken or stationary). Validamycin A caused a reduction in the number and viability of conidia produced by F. culmorum.

Antifungal Agents↗

PubMed1987

Glucose and penicillin concentrations in agar medium below fungal colonies.

The growth of colonies of Rhizoctonia cerealis and Penicillium chrysogenum on solid media in plate cultures was studied. When grown on defined media containing 10-50 mM-glucose, R. cerealis did not cause a significant reduction in the glucose concentration of the medium in advance of colonization, but did cause the formation of a steep glucose concentration gradient in the substrate below the colony; the medium directly below the centre of a 7 cm diameter colony of R. cerealis was exhausted of glucose even when the fungus was grown on medium containing 50 mM-glucose. Penicillin produced by colonies of P. chrysogenum accumulated in the medium in advance of fungal colonization. For a period up to about 18 d after inoculation, the concentration of penicillin in the medium throughout the plate increased with colony development and thereafter, except at the margins of the plate, decreased.

Adenosine Triphosphate↗