Removal of Cr(VI) from ground water by Saccharomyces cerevisiae

Chromium can be removed from ground water by the unicellular yeast, Saccharomyces cerevisiae. Local ground water maintains chromium as CrO₄ ^2- because of bicarbonate buffering and pH and E _h conditions (8.2 and +343 mV, respectively). In laboratory studies, we used commercially available, nonpathogenic S. cerevisiae to remove hexavalent chromium [Cr(VI)] from ground water. The influence of parameters such as temperature, pH, and glucose concentration on Cr(VI) removal by yeast were also examined. S. cerevisiae removed Cr(VI) under aerobic and anaerobic conditions, with a slightly greater rate occurring under anaerobic conditions. Our kinetic studies reveal a reaction rate (V_max) of 0.227 mg h^-1 (g dry wt biomass)^-1 and a Michaelis constant (Km) of 145 mg/l in natural ground water using mature S. cerevisiae cultures. We found a rapid (within 2 minutes) initial removal of Cr(VI) with freshly hydrated cells [55–67 mg h^-1 (g dry wt biomass)^-1] followed by a much slower uptake [0.6–1.1 mg h^-1 (g dry wt biomass)^-1] that diminished with time. A materials-balance for a batch reactor over 24 hours resulted in an overall shift in redox potential from +321 to +90 mV, an increase in the bicarbonate concentration (150–3400 mg/l) and a decrease in the Cr(VI) concentration in the effluent (1.9-0 mg/l).     

Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae.

Bleomycin mediates cell wall damage in the yeast Saccharomyces cerevisiae. Bleomycin treatments in the presence of Fe(II) increased the rate of spheroplast formation by lytic enzymes by 5- to 40-fold. Neither Fe(III) nor other tested ions caused significant cell wall damage in the presence of bleomycin. The effect of bleomycin-Fe(II) on the cell wall mimicked the characteristics of bleomycin-Fe(II)-mediated DNA damage in dependence on aeration, inhibition by ascorbate, and potentiation by submillimolar concentrations of sodium phosphate. Bleomycin-mediated cell wall damage was time and dose dependent, with incubations as short as 20 min and drug concentrations as low as 3.3 x 10(-7)M causing measurable cell wall damage in strain CM1069-40. These times and concentrations are within the range of effectiveness for bleomycin-mediated DNA damage and for the cytotoxicity of the drug. Although Fe(III) was inactive with bleomycin and O2, the bleomycin-Fe(III) complex damaged walls and lysed cells in the presence of H2O2. H2O2 causes similar activation of bleomycin-Fe(III) in assays of DNA scission. These results suggest that an activated bleomycin-Fe-O2 complex disrupts essential cell wall polymers in a manner analogous to bleomycin-mediated cleavage of DNA.     

Ycf1p-dependent Hg(II) detoxification in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, disruption of the YCF1 gene increases the sensitivity of cell growth to mercury. Transformation of the resulting ycf1 null mutant with a plasmid harbouring YCF1 under the control of the GAL promoter largely restores the wild-type resistance to the metal ion. The protective effect of Ycf1p against the toxicity of mercury is especially pronounced when yeast cells are grown in rich medium or in minimal medium supplemented with glutathione. Secretory vesicles from S. cerevisiae cells overproducing Ycf1p are shown to exhibit ATP-dependent transport of bis(glutathionato)mercury. Moreover, using beta-galactosidase as a reporter protein, a relationship between mercury addition and the activity of the YCF1 promoter can be shown. Altogether, these observations indicate a defence mechanism involving an induction of the expression of Ycf1p and transport by this protein of mercury-glutathione adducts into the vacuole. Finally, possible coparticipation in mercury tolerance of other ABC proteins sharing close homology with Ycf1p was investigated. Gene disruption experiments enable us to conclude that neither Bpt1p, Yor1p, Ybt1p nor YHL035p plays a major role in the detoxification of mercury.     

Removal of Frameshift Intermediates by Mismatch Repair Proteins in Saccharomyces cerevisiae

Frameshift mutations occur when the coding region of a gene is altered by addition or deletion of a number of base pairs that is not a multiple of three. The occurrence of a deletion versus an insertion type of frameshift depends on the nature of the transient intermediate structure formed during DNA synthesis. Extrahelical bases on the template strand give rise to deletions, whereas extrahelical bases on the strand being synthesized produce insertions. We previously used reversion of a +1 frameshift mutation to analyze the role of the mismatch repair (MMR) machinery in correcting -1 frameshift intermediates within a defined region of the yeast LYS2 gene. In this study, we have used reversion of a -1 frameshift mutation within the same region of LYS2 to analyze the role of the MMR machinery in the correction of frameshift intermediates that give rise to insertion events. We found that insertion and deletion events occur at similar rates but that the reversion spectra are very different in both the wild-type and MMR-defective backgrounds. In addition, analysis of the +1 spectra revealed novel roles for Msh3p and Msh6p in removing specific types of frameshift intermediates.     

Response of Saccharomyces cerevisiae to lead ion stress

The response of Saccharomyces cerevisiae to different concentrations of Pb²⁺ was investigated. The results demonstrated that the growth of S. cerevisiae in the presence of Pb²⁺ showed a lag phase much longer than that in the absence of Pb²⁺. The inhibition was dependent upon Pb²⁺ concentrations. The Pb²⁺ at a concentration of 5 μM inhibited the microbial growth by approximately 30% with regard to control, whereas Pb²⁺ at concentration of 2 μM did not have a significant effect on the microbial growth. The existence of Pb²⁺ did not perturb cell-protein synthesis and there was a good correlation between dry cell weights and total protein content (R ² = 0.98). The RNA/DNA ratio in the microbial cells varied with Pb²⁺ concentration and there was a significant positive correlation between Pb²⁺ concentration and the RNA/DNA ratio. The microbial assimilation of ammonium ion was inhibited by the presence of Pb²⁺ in the medium; when Pb²⁺ concentration was 10 μM, the microbial ammonium assimilation was inhibited about 50%, in comparison with the control experiment.     

Fixation of spent Saccharomyces cerevisiae biomass for lead sorption

Spent Saccharomyces cerevisiae cells from a beer fermentation process were evaluated for lead cation sorption. The crude biomass was washed with water and acetone prior to any other treatment. Although the washed biomass showed substantial lead ion sorption it was susceptible to microbial spoilage. Different aldehydes were tested as chemical fixation agents; however, most of them caused drastic lowering of the metal uptake capacity. However, benzaldehyde was not only an excellent fixation agent, but the biomass treated with it also retained its original lead sorption capacity. A mechanism for the fixation process is suggested. Received: 11 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

Sucrose inversion by gelatin-entrapped cells of yeast (Saccharomyces cerevisiae)

Summary Sucrose hydrolysis by invertase-active yeast cells (S. cerevisiae) entrapped in gelatin was investigated using different types of miniaturized reactors. The entrapped preparations showed the highest operational stability in a continuous stirred-tank reactor. The invertase activity of the entrapped preparation was found to be almost independent of the buffer concentration so that sucrose invermay be conducted in a non-buffered medium.     

Biosynthesis of L-ascorbic acid (vitamin C) by Saccharomyces cerevisiae

Saccharomyces cerevisiae cells incubated with D-glucose (D-Glc), D-galactose or D-mannose (D-Man) synthesised D-erythroascorbic acid (D-EAA) but not L-ascorbic acid (L-AA). Accumulation of D-EAA was observed in cells incubated with D-arabinose (D-Ara) whilst accumulation of L-AA occurred in cells incubated with L-galactose (L-Gal), L-galactono-1,4-lactone and L-gulono-1,4-lactone. When S. cerevisiae cells were incubated with D-[U-(14)C]Glc, D-[U-(14)C]Man or L-[1-(14)C]Gal, incorporation of radioactivity into L-AA was observed only with L-[1-(14)C]Gal. Pre-incubation of yeast cells with D-Ara substantially reduced the incorporation of L-[1-(14)C]Gal into L-AA. Our results indicate that, under appropriate conditions, yeast cells can synthesise L-AA via the pathway naturally used for D-EAA biosynthesis.     

Specificity of yeast (Saccharomyces cerevisiae) in removing carbohydrates by fermentation

The specificity of Saccharomyces cerevisiae yeast on the removal of carbohydrates by fermentation was studied. The common monosaccharides, D-glucose, D-fructose, D-mannose, and D-galactose were completely removed; D-glucuronic acid and D-ribose were partially removed; but D-xylose, D-rhamnose, and L-sorbose were not removed and were completely resistant. Of four glycosides, methyl and phenyl alpha- and beta-D-glucopyranosides, three of the four were partially removed and methyl beta-D-glucopyranoside was not removed. The disaccharides, maltose, sucrose, and turanose were completely removed, while cellobiose, lactose, and melibiose were completely resistant. Isomaltose and alpha,alpha-trehalose were partially removed. Maltotriose and raffinose were partially removed, but isomaltotriose and melezitose were completely resistant. The tetrasaccharides, maltotetraose, isomaltotetraose, and acarbose, were completely resistant. Further, the yeast enzymes did not alter any of the resistant carbohydrates by transglycosylation or condensation reactions or by any other types of reactions.     

D-tyrosyl-tRNA(Tyr) metabolism in Saccharomyces cerevisiae

The Saccharomyces cerevisiae YDL219w (DTD1) gene, which codes for an amino acid sequence sharing 34% identity with the Escherichia coli D-Tyr-tRNA(Tyr) deacylase, was cloned, and its product was functionally characterized. Overexpression in the yeast of the DTD1 gene from a multicopy plasmid increased D-Tyr-tRNA(Tyr) deacylase activity in crude extracts by two orders of magnitude. Upon disruption of the chromosomal gene, deacylase activity was decreased by more than 90%, and the sensitivity to D-tyrosine of the growth of S. cerevisiae was exacerbated. The toxicity of D-tyrosine was also enhanced under conditions of nitrogen starvation, which stimulate the uptake of D-amino acids. In relation with these behaviors, the capacity of purified S. cerevisiae tyrosyl-tRNA synthetase to produce D-Tyr-tRNA(Tyr) could be shown. Finally, the phylogenetic distribution of genes homologous to DTD1 was examined in connection with L-tyrosine prototrophy or auxotrophy. In the auxotrophs, DTD1-like genes are systematically absent. In the prototrophs, the putative occurrence of a deacylase is variable. It possibly depends on the L-tyrosine anabolic pathway adopted by the cell.     

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

Summary When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the nitrogen source, significant amounts of malate were formed when the pH was kept constant by buffering. Trace metals had no or only little influence on malate synthesis. Biotin and pantothenate were essential for growth. Added ¹⁴CO₂ led to the formation of approximately equal quantities of labelled malate and succinate by S. cerevisiae strain 52, whereas about ten times more malate than succinate was formed by Saccharomyces uvarum. Avidin strongly inhibited the formation of malate while the inhibiton of succinate synthesis and of growth was comparatively much less. Malate is obviously formed by reduction of oxalacetate, the synthesis of which is catalysed by a biotin-dependent pyruvate carboxylase.     

Sequestration of arginine by polyphosphate in vacuoles of yeast (Saccharomyces cerevisiae)

The conditions for synthesis, purification, and properties of tryptophanase by a marine organism (Vibrio K-7) were studied. Tryptophanase was induced by tryptophan and its analogs, and partially repressed by 0.5% glucose or glycerol. NaCl (0.4M) was required for optimal growth and tryptophanase activity in whole cells. The enzyme was purified to 92% homogeneity by heat treatment, hydroxyapatite chromatography and fractionation with ammonium sulfate. This tryptophanase has been found to have kinetic properties similar to the tryptophanase from other microorganisms. It carries out both , -elimination reactions (using tryptophan, serine, cysteine and S-methyl-cysteine as substrates) and -replacement reactions (forming tryptophan from indole and serine, cysteine or S-methyl-cysteine). The enzyme has a sedimentation coefficient of 9.2S and requires pyridoxal 5-phosphate as a cofactor. The optimal pH for the tryptophanase reaction is pH 8.0.Nonstandard Abbreviations PLP pyridoxal 5-phosphate - TPase tryptophanase - TSase tryptophan synthase - DHase dehydratase - TCA tricarboxylic acid - BSA bovine serum albumin Preliminary reports of this work have been presented (M. J. Klug and R. D. DeMoss, Bacteriol. Proc. 1971, p. 132; D. D. Whitt and R. D. DeMoss, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, p. 148)     

ESR study of copper(II) retention by entire cell, cells walls, and protoplasts of Saccharomyces cerevisiae

Copper retention by whole cells, protoplasts, and isolated cell walls of Saccharomyces cerevisiae was investigated in the absence of any energy source in the medium. The cell walls accounted only for a small fraction of the cation retention by whole cells. ESR results showed that copper was not bound only at the outer face of the plasma membrane, but it was also distributed in the plasma membrane and (or) in the cytoplasm. ESR studies also showed that, in all three systems, copper was chelated by peptides or proteins. The binding sites were formed by an amide and a strongly complexing ligand such as an amine. Their configuration depended upon pH: in slightly acidic conditions, copper was bound by the oxygen of the amide; at basic pH, NHCO became deprotonated and the negatively charged nitrogen bound to the metal.     

Activation of serine sulphydrase from baker's yeast (Saccharomyces cerevisiae) by vanadate

Vanadate stimulates the liberation of H2S from cysteine in intact cells of baker's yeast (Saccharomyces cerevisiae) with a maximal increase of 60% at 10 microM NH4VO3. Protein separation from crude yeast extract yielded two active protein fractions which were found to catalyze the degradation of cysteine to H2S, pyruvate and ammonia or H2S and serine, respectively, thus characterizing them as cysteine desulphydrase and serine sulphydrase. Only the latter enzyme was found to be activated by vanadate, showing optimal enhancement of about 100% at 10 microM NH4VO3.     

Secretive expression of the Aspergillus aculeatus cellulase (FI-CMCase) by Saccharomyces cerevisiae

A cDNA encoding FI-carboxymethylcellulase (FI-CMCase) of the fungus Aspergillus aculeatus was expressed in Saccharomyces cerevisiae under the control of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) promoter of S. cerevisiae. The transformed cells were able to secrete FI-CMCase efficiently into the culture medium as active enzyme. The recombinant FI-CMCases were observed to be two different enzymes of different molecular mass, one of which corresponded to native FI-CMCase (non-glycosylated FI-CMCase) and the other of which was an N-glycosylated protein (glycosylated FI-CMCase). The recombinant glycosylated FI-CMCase showed a higher thermostability than that of the native enzyme, although the former showed slightly lower activity toward the substrate than the latter.     

Selenomethionine incorporation in Saccharomyces cerevisiae RNA polymerase II

A protocol for the incorporation of SeMet into yeast proteins is described. Incorporation at a level of about 50% suffices for the location of Se sites in an anomalous difference Fourier map of the 0.5 MDa yeast RNA polymerase II. This shows the utility of the approach as an aid in the model-building of large protein complexes.     

Continuous fixed-bed column study and adsorption modeling: Removal of cadmium (II) and lead (II) ions in aqueous solution by dead calcareous skeletons

The sorption of Cd(II) and Pb(II) ions was conducted in a continuous fixed-bed column by using dead calcareous skeletons (CS). The column performances were evaluated by varying the adsorbent bed height, influent flow rate and metals initial concentration. The breakthrough curve for the bed height indicated that a longer bed column prolonged the life span of the column with a maximum capacity of 26.447 and 38.460 mg/g for the Cd(II) and Pb(II) column, respectively. The increased flow rate and initial concentration caused the column exhaustion time to occur earlier. The experimental column data were also expressed in column adsorption models, namely, the Thomas, Yoon–Nelson and Adam–Bohart models. The Thomas model fitted well with the Cd(II) data with the correlated curve (r² > 0.9). The Yoon–Nelson model was selected to predict the 50% breakthrough time achieved by the column system and provided the estimated breakthrough time for the columns that were not exhausted during the operation. The Adam–Bohart model was applicable for the initial part of adsorption with the saturation concentration data at the equilibrium. The saturation index of aragonite and calcite depicted that dissolution of calcium occurred in the aqueous solution. The experimental and theoretical data were correlated with a significant relationship trend (p < 0.01), which showed that the trend of experimental data fit well with the modeling trend. The trends of both the experimental and theoretical data were strongly and significantly correlated due to involving the column parameters and the components of CS.