Binding of substrates and other anions to yeast phosphoglycerate kinase.

1. The binding of all four substrates to yeast phosphoglycerate kinase has been studied using a gel filtration technique. The binding of phosphate and sulphate anions has also been investigated. 2. Two sites for each adenine nucleotide were found, one site being weaker than the other by between 30 and 50-fold. Only one binding site for the phosphoglycerate substrates was found. 3. 1,3-Bisphosphoglycerate (1,3-P2-glycerate) bound to the enzyme approximately 1000 times tighter than the other three substrates, its dissociation constant being 0.06 micrometer at ionic strength 0.15 M. 4. Sulphate and phosphate were mutually competitive and sulphate competed with the binding of all substrates except MgADP. MgADP bound to the enzyme more weakly in the presence of sulphate. The dissociation constant for sulphate binding was 1.6 mM at ionic strength of 0.15 M, and 0.05 mM at ionic strength 0.015 M. 5. These results are consistent with sulphate acting as a competitive inhibitor, as found by kinetic studies at high sulphate concentrations. The activatory effect of sulphate at lower concentrations and the substrate activation phenomea displayed by this enzyme, are interpreted in terms of a two-step dissociation of 1, 3-P2-glycerate. The presence of moderate concentrations of MgATP, 3-phosphoglycerate or sulphate causes acceleration of the rate of dissociation of the product, 1, 3-P2-glycerate, this being the rate-limiting step in the overall enzyme reaction.     

Effect of utilization of substrates on the composition of dental plaque

Abstract The microbiota in the mouth is subjected to substrate limitations. In this study we have evaluated the role of competition for carbon and energy substrates on the proportions of 2 microbial species in a simplified plaque ecosystem in gnotobiotic rats. Germ-free rats were inoculated with a combination of Streptococcus sanguis and Streptococcus mutans , or with a combination of Streptococcus milleri and S. mutans . The available carbon and energy sources were varied through the host's diet. 3 Experimental diets were tested: (i) a basal diet low in soluble carbohydrates; (ii) an arginine-supplemented diet; (iii) a sucrose-supplemented diet. Arginine is used for growth by S. sanguis and S. milleri , but not by S. mutans . Sucrose is rapidly fermented by all 3 species.
The total number of viable organisms on the dentition increased when arginine or sucrose were supplied in the diet. With the arginine-supplemented diet, S. sanguis and S. milleri increased while S. mutans decreased. With the sucrose-supplemented diet, S. mutans increased while S. sanguis and S. milleri decreased. These results were explained by assuming that the organism with the highest growth rate on the supplementary substrate competes most favourably. Changes in the environmental pH, due to breakdown of sucrose and arginine, might also have affected the competition between the streptococci. In addition, production of extracellular glucans from sucrose could be a competitive advantage for S. mutans .     

Effect of nystatin on the release of glycerol from salt-stressed cells of the salt-tolerant yeast Zygosaccharomyces rouxii

The polyene antibiotic nystatin, which affects fungal membrane permeability, inhibited the growth of Zygosaccharomyces rouxii grown in medium containing 15% (w/v) NaCl, whereas yeast grown in medium without NaCl were only slightly inhibited. Nystatin caused salt-stressed cells to release large amounts of glycerol and inhibited their growth, but amino acids and materials with an absorbance at 260 nm were not released from the cells. The leakage was increased by the addition of glucose, and more than 90% of the intracellular glycerol was released into the medium during a 2-h incubation with 0.11 microM nystatin and 2% (w/v) glucose. Glycerol was indispensable for the growth of Z. rouxii grown in culture medium containing 15% NaCl.     

Temperature relationships of fungi isolated at low temperatures from soils and other substrates

A study of the temperature relationships of 20 mycelial and yeast fungi which had been isolated at low temperatures from soils and from abattoirs indicated that few fungi can be regarded as truly psychrophilic. Only 1 species failed to grow at 25C. Although all the species investigated were able to grow at 4C and can therefore be considered as potential spoilage organisms on refrigerated foods, their optimal growth temperature was either 15C or 25C. The 20 species could be divided into 4 groups in relation to their temperature relationships, particularly their optimal temperatures and their ability to grow at 30C.A number of fungi able to grow at 4C has been isolated from soils and from abattoirs in New Zealand (1, 2). Several mycelial fungi can show reasonable growth below 10C, and some even down to –10C, but, when laboratory studies have been performed, most of these fungi prove to have an optimal growth temperature at 25C or above (3). These fungi, however, can be a cause of serious spoilage of a variety of refrigerated foods and even at the temperature currently used for meat storage ( –12C), occasional consignments of meat are encountered which show the characteristic spots of fungal contamination. During investigations into the occurrence of these fungi in an abattoir, (1), 9 mould and 4 yeast species were isolated which could grow at 4C, and further studies into the reservoirs of these fungi in the outside environment resulted in the isolation of a further 8 mould and 2 yeast species (2). This paper reports on investigations into the temperature relationships of 20 of these species.     

Engineering of glycosylation in yeast and other fungi: current state and perspectives

With the increasing demand for recombinant proteins and glycoproteins, research on hosts for producing these proteins is focusing increasingly on more cost-effective expression systems. Yeasts and other fungi are promising alternatives because they provide easy and cheap systems that can perform eukaryotic post-translational modifications. Unfortunately, yeasts and other fungi modify their glycoproteins with heterogeneous high-mannose glycan structures, which is often detrimental to a therapeutic protein’s pharmacokinetic behavior and can reduce the efficiency of downstream processing. This problem can be solved by engineering the glycosylation pathways to produce homogeneous and, if so desired, human-like glycan structures. In this review, we provide an overview of the most significant recently reported approaches for engineering the glycosylation pathways in yeasts and fungi.     

Biomass protein formation by filamentous fungi on woody substrates

Summary Growth and biomass protein formation by filamentous fungi grown on pretreated tropical woods of Mesta (Hibiscus cannabinus Linn.) and Subabul [Leucaena leucocephala (Lam.) de Witt] as well as their isolated hemicellulose and cellulose fractions have been studied. Penicillium janthinellum and Penicillium funiculosum produced a biomass having 20 to 30% crude protein when grown on either hemicellulose, while growth on pretreated (autoclaved in 1% NaOH) wood or isolated cellulose fractions was comparatively poor and crude protein content only 5 to 8% in the biomass.NCL Communication no.3550     

Crude oil utilization by fungi.

Sixty fungal isolates, 34 obtained by a static enrichment technique from soils of northern Canadian oil-producing areas and 26 from culture collections, were screened for their ability to grow on n-tetradecane, toluene, naphthalene, and seven crude oils of varying composition. Forty cultures, including 28 soil isolates, were capable of growth on one or more crude oils. The genera most frequently isolated from soils were those producing abundant small condida, e.g. Penicillium and Verticillium spp. Oil-degrading strains of Beauveria bassiana, Mortieriella sp., Phoma sp., Scolecobasidium obovatum, and Tolypocladium inflatum were also isolated. Qualitative and quantitative differences were noted among the capacities of different crude oils to sustain the growth of individual fungal isolates. Data are presented which show that ability to grow on a pure n-alkane is not a good indicator of ability to grow on crude oil. Degradation of Rainbow Lake crude oil by individual isolates was demonstrated by gravimetric and gas-chromatographic techniques. The problems involved in determining the response and the potential of fungi to degrade oil spilled in the environment are discussed.     

Effect of volatiles from bacteria and yeast on the growth and pigmentation of sapstain fungi

Sapstain fungi affect the appearance of wood due to colonisation by pigmented hyphae but without producing significant strength losses. This is due to the production of melanin in the fungal cell walls of the staining fungi. Any biological control strategy targeted against this type of deterioration would therefore be considered successful if it inhibited either fungal growth or pigment production. Previous work has established that specific bacterial and yeast isolates selected on the basis of agar screening studies could significantly reduce levels of staining in wood block tests. This paper presents the results of a study to examine the role of volatile organic compounds (VOCs) produced by four bacterial and three yeast isolates on the growth and pigment production by a range of five sapstain fungi on three media types. VOCs from three of the four bacterial strains tested completely inhibited growth of the five target sapstain fungi but only when the antagonists were grown on tryptone soya media. When antagonists were grown on either malt agar or a low nutrient medium levels of inhibition were either significantly reduced or non-existent. Yeast antagonists generally produced lower levels of growth inhibition than the bacteria but a Williopsis mrakii isolate gave 100% inhibition of three of the five sapstain fungi. Production of inhibitory VOCs was highly dependent on the specific antagonist as well as its growth substrate and all five sapstain fungi showed varying sensitivities to the VOCs produced. Not all fungi were inhibited, growth of O. piliferum and A. pullulans being stimulated by the VOCs from antagonists but only when grown under low nutrient conditions. In some instances, where growth was only slightly reduced, the level of pigmentation of the sapstain colony was significantly reduced compared with corresponding controls. The implications of this work for the biological control of sapstain fungi are discussed.     

Effect of carbon levels from three organic substrates on egyptian soil fungi

Summary The fungal composition of soils with an equivalent percentage of carbon levels 2%, 5%, 10% and 20% fromOlea europea,, Citrus sinensis andEucalyptus rostrato leaves was studied after 10, 45 and 120 days using the dilution-plate method. They all induced significant increases in the total count of fungi, but their effect on the individual fungi was selective. The best substrate for the total fungus flora wasCitrus sinensis and the poorest wasOlea europeae leaves. The best three colonisers (possessing the highest counts), at carbon levels 2%, 5%, 10% and 20% at the end of the experimental periods were, respectively, as follows: forOlea europeae leaves,Scopulariaopsis brevicaulis, Drechslera halodes andCylindrocarpon sp.;Aspergillus quadrilineatus, A. sydowi andCurvularia lunata; A. quadrilineatus, Alternaria alternata andC. lunata andA. quadrilineatus, A. egyptiacus andA. terreus; forCitrus sinensis leaves,A. quadrilineatus, A. fumigatus andS. brumptii; A. fumigatus, A. quadrilineatus andA. versicolor; A. quadrilineatus, A. terreus andS. brumptii; andA. quadrilineatus, A. flavus andA. sydowi; and forEucalyptus rostrata leaves,A. quadrilineatus, Rhizopus stolonifer andA. sydowi; A. quadrilineatus, A. niger andA. terreus; A. niger, A. quadrilineatus andS. brevicaulis; andA. terreus, A. niger andA. quadrilineatus.     

Influence of glucose and other substrates on electric field and polyethylene glycol-mediated transformation of intact yeast cells

Abstract A prepulse incubation with 2% glucose (mannose, fructose) strongly inhibited electrotransformation of intact yeast cells. This inhibitory effect was not due to alterations of cell viability or to cell membrane electropermeabilization, and was not affected by the solution buffer properties of preincubation and pulsing media. The electrotransformation efficiency was not modified by non-metabolized substances, such as sorbitol or alose. The glucose inhibition of electrotransformation was fast, occurring after only a 3–5-min preincubation. The postpulse incubation with substrate also decreased transformation efficiency, but was pH-dependent. We observed a similar, strong inhibitory effect of glucose when cells were transformed chemically. A pH dependence of yeast electrotransformation was established.     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

Zn biosorption by Rhizopus arrhizus and other fungi

Biosorption of zinc ions by inactivated fungal mycelia was studied. Of the six fungal species, Rhizopus arrhizus, Mucor racemosus, Mycotypha africana, Aspergillus nidulans, Aspergillus niger and Schizosaccharomyces pombe, R. arrhizus exhibited the highest capacity (Q ^max = 213 μmol g⁻¹ dry weight). Further experiments with different cellular fractions of R. arrhizus showed that Zn was predominantly bound to cell-wall chitin and chitosan (Q ^max = 312 μmol g⁻¹ dry weight). Adsorption data were best modelled by the Langmuir isotherm, although they can be modelled by the Freundlich equation as well at relatively low aqueous concentrations. Biosorption generally decreased with increase in biosorbent particle size and its concentration. Low pH reduced Zn sorption, because of the strong competition from hydrogen ions for binding sites on fungi. The presence of ligands reduced metal uptake, chiefly by forming metal complexes of a less biosorbable nature. Received: 2 November 1998 / Received revision: 12 January 1999 / Accepted: 17 January 1999     

Effects of inhibition and repression on the utilization of substrates by heterogeneous bacterial communities

This investigation attempts to evaluate to what extent enzyme inhibition and repression by metabolites, indigenous to the cell, are significant phenomena in natural microbial communities. Three case histories of the kinetics of substrate utilization and growth in multisubstrate media by heterogeneous bacterial populations are presented: (i) concurrent substrate utilization and growth on both substrates simultaneously (glucose plus benzoate); (ii) sequential substrate elimination accompanied by diauxic growth as a result of inhibition of enzyme activity (glucose plus galactose); (iii) sequential substrate utilization accompanied by diauxic growth caused by repression of enzyme formation (glucose plus l-phenylalanine, benzoate plus l-phenylalanine). It is shown that enzyme inhibition was observed in two-substrate media as well as in multisubstrate media and was maintained at low substrate concentrations (few milligrams per liter). A special attempt has been made to maintain the diversity of the experimental microbial population during the adaptation and enrichment period. All substrates were determined with sensitive analytical methods specific for the individual substrates. The results obtained confirm that catabolite repression and the resulting sequential substrate utilization are observed in heterogeneous bacterial populations.