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 .     

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.     

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.     

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.     

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.     

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.     

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     

Simultaneous utilization of glucose and sucrose by thermophilic fungi.

The utilization of mixtures of glucose and sucrose at nonlimiting concentrations was studied in batch cultures of two common thermophilic fungi, Thermomyces lanuginosus and Penicilium duponti. The sucrose-utilizing enzymes (sucrose permease and invertase) in both fungi were inducible. Both sugars were used concurrently, regardless of their relative proportion in the mixture. At the optimal growth temperature (50 degrees C), T. lanuginosus utilized sucrose earlier than it did glucose, but at a suboptimal growth temperature (30 degrees C) the two sugars were utilized at nearly comparable rates. The coutilization of the two sugars was most likely possible because (i) invertase was insensitive to catabolite repression by glucose, (ii) the activity and affinity of the glucose transport system were lowered when sucrose was included in the growth medium, and (iii) the activity of the glucose uptake system was also subject to repression by high concentrations of glucose itself. The concurrent utilization of the available carbon sources by thermophilic fungi might be an adaptive strategy for opportunistic growth in nature under conditions of low nutrient availability and thermal fluctuations in the environment.     

Steroid transformations by species of Cephalosporium and other fungi

A total of 58 cultures, tentatively identified as species of the genus Cephalosporium, were screened in flask fermentations for their ability to effect conversions of progesterone (Delta(4)-pregnene-3,20-dione) and Reichstein's Substance S (Delta(4)-pregnene-17alpha,21-diol-3,20-dione). A large number of transformations were observed by means of a series of five paper chromatography systems rated for analysis of steroid compounds ranging in polarity from progesterone to polyhydroxylated steroids. Five different transformation products were selected for isolation and identification. For purposes of recovery, conversions were conducted under submerged conditions in either 4- or 200-liter fermentors in which the broth was agitated and aerated. The steroid substrate was dissolved in acetone and added aseptically to the growing culture in a final concentration of 0.025%. After the conversions were effected, the whole broth was extracted with chloroform, and the transformation products were recovered, either by direct crystallization from solvents or through the use of silica gel columns. It was determined that C. ciferrii 21C converted progesterone to Delta(4)-androstene-3,17-dione. Kendall's Compound F (Delta(4)-pregnene-11beta,17alpha,21-triol-3,20-dione) was converted to its 20beta-ol analogue by Geotrichum sp. 51C (during these studies, a number of cultures were taxonomically reclassified). Cephalosporium sp. 27C formed the Delta(1)-analogue of Reichstein's Substance S, and Cephalosporium sclerotigenum 31C and Verticillium aphidum both converted Substance S to the 6beta-hydroxy derivative. Paecilomyces persicinus 22C converted Substance S to a product believed to be a dihydroxylated derivative.