The use of solid medium to study effects of cadmium, copper and zinc on yeasts and yeast-like fungi: applicability and limitations

A defined solid medium has been used to examine the responses of Aureobasidium pullulans, Saccharomyces cerevisiae and Sporobolomyces roseus to cadmium, copper and zinc. Experiments where aliquot volumes of metal salt solutions were added to wells in the centre of agar plates revealed marked differences between these organisms. The yeast-like fungus A. pullulans was the least sensitive but S. cerevisiae was more sensitive to cadmium than zinc: the reverse was true for S. roseus. Quantitative data, which complemented the qualitative results, were obtained by measurement of metal concentrations in the plates.     

Production of antibacterial compounds by phylloplane-inhabiting yeasts and yeastlike fungi.

The production of antibacterial compounds by yeasts and yeastlike fungi isolated from the phylloplane is reported. Aureobasidium pullulans, Citeromyces matritensis, Cryptococcus laurentii, Rhodotorula glutinis, and Sporobolomyces roseus produced antibacterial compounds inhibitory to both Pseudomonas fluorescens and Staphylococcus aureus in an overlay bioassay. In contrast, isolates of Candida albicans, Filobasidium uniguttulatum, Saccharomyces cerevisiae, Torulaspora delbruckii, Tremella foliacea, Trichosporon beigelii, and Trichosporon dulcitum obtained from soil or from culture collections did not produce inhibitory compounds when screened by the same procedure. The production of antibacterial compounds was examined in more detail, using several isolates of A. pullulans distinguished by cluster analysis on the basis of biochemical and physiological tests. They were found to produce a range of antibacterial compounds with different activities. Two distinct antibiotics were produced by an isolate of A. pullulans in liquid culture during both the logarithmic and the stationary phases of growth.     

Effects of heavy metal pollution on the microflora of pine needles

A leaf washing method was used to compare the numbers and species composition of the phylloplane microflora of corsican pine saplings (a) uncontaminated, (b) exposed to zinc, lead and cadmium pollution from a smelter and (c) artificially dosed with zinc, lead and cadmium oxides to simulate the emissions from the smelter without the complicating effects of other pollutants. There was little difference in total numbers of most groups of micro-organisms isolated from each site, although both sets of metal-contaminated saplings generally supported a higher percentage of metal tolerant isolates than the uncontaminated plants. Artificially dosed plants also supported greater numbers of Aureobasidium pullulans and orange and pink chromogenic bacteria. These particular organisms were also among the most metal-tolerant fungi and bacteria in vitro.     

Role of Competition for Sugars by Yeasts in the Biocontrol of Gray Mold of Apple

The yeasts, Cryptococcus laurentii BSR-Y22 or Sporobolomyces roseus FS-43-238, but not Saccharomyces cerevisiae BY-1, reduced gray mold ( Botrytis cinerea ) when applied to wounds of apples (cv. Golden Delicious) which were stored at 22IC for 7 days or 3IC for up to 2 months. The role of competition for sugars by these yeasts as a mechanism of antagonism was investigated at 22IC. Populations of C. laurentii and S. roseus in wounds were 6-9 times greater than those of S. cerevisiae from 1 to 7 days following inoculation. Yeasts in wounds utilized more 14C-labelled fructose, glucose or sucrose than conidia of B. cinerea during 48 h, but yeasts did not differ in their utilization of sugars. Utilization of 14C-sugars by yeasts in vitro was greater at most sampling times for conidia; the uptake after 48 h was always greater for yeasts and the addition of nitrogen did not alter this result. The utilization of 14C-sugars by S. roseus in vitro was greater than that in the other yeasts. The uptake and utilization of 14C-fructose by C. laurentii or S. roseus was greater than that of S. cerevisiae , but the utilization of glucose or sucrose by C. laurentii and S. cerevisiae was similar and the uptake of these sugars by these yeasts did not differ. Yeasts mixed with conidia in sterile, dilute solutions of fructose, glucose or sucrose, or in dilute apple juice inhibited conidial germination compared with no-yeast controls; S. cerevisiae was less effective than C. laurentii or S. roseus . Only yeasts rapidly depleted sugars from juice or sugar solutions. Yeasts did not alter the pH or oxygen content of dilute juice to the detriment of conidial germination. These results strongly suggest that competition for sugars by yeasts played a role in the biocontrol of gray mold, but that some other factor(s) most likely contributed to differences in efficacy between the yeasts.     

Comparison of Pb2 accumulation characteristics between live and dead cells of Saccharomyces cerevisiae and Aureobasidium pullulans

Pb2+ accumulation processes between live and dead cells of Saccharomyces cerevisiae and Aureobasidium pullulans are different. In the case of S. cerevisiae, the Pb2+ accumulation capacity of the live cells was higher than that of the dead cells but they showed reversed initial Pb2+ accumulation rates. On the contrary, A. pullulans used a different process due to the existence of extracellular polymeric substances, which allowed both the capacity and the initial rate of Pb2+ accumulation in the live cells to be higher than those in the dead cells. © Rapid Science Ltd. 1998     

Heavy metal composition of polysomal fractions following cadmium challenge

Endogeneous levels of zinc and copper were found to be 1.2±0.1×10⁻² and 0.3±0.1×10⁻² μg/A260 unit, respectively, in polysomal fractions from control animals; cadmium, however, was undetectable. In experimental animals (injected with cadmium) zinc, copper, and cadmium were found in polysomal fractions isolated by two different methods. One hour after a cadmium injection there was a rise in both the zinc and copper content of the polysomal fractions, which then declined steadily to below control levels by 16 h. Neither zinc nor cadmium were dialyzable from these fractions by a TRIS buffer; however, addition of 0.01M EDTA to the buffer resulted in removal of 75% of the zinc and all of the detectable cadmium. The addition of cadmium (CdCl₂) to control supernatants (adjusted to the cadmium concentration present in supernatants 6 h after in vivo exposure) resulted in metal binding to polysomal fractions in levels comparable to those observed after in vivo exposures to the metal. When cadmium was added in the form of cadmium thionein, a smaller fraction of the metal was isolated with the polysomal fraction. Cadmium bound to polysomal fractions in vivo (24 h after exposure) was sensitive to release by protease digestion, but insensitive to release by ribonuclease digestion.     

The fate of carboxin, benomyl and thiabendazole in seed- and soil-treated plants, as shown by in vitro and in vivo bioassays on some epiphytic yeasts

The yeast, Sporobolomyces roseus, when added to leaves of barley and pea seedlings seed- and soil-treated with carboxin, benomyl or thiabendazole (TBZ), sporulated little or not at all from both sides of the first leaves. Second leaves of barley and third of pea gave similar results. Tests in vivo showed that all three chemicals reduced sporulation of S. roseus at lower concentrations than they did growth, and also delayed sporulation. Leaves from benomyl- or TBZ-treated plants inhibited the growth of sensitive test organisms (S. roseus, Leucosporidium scottii and Aureobasidium pullulans), on agar: it is considered that fungitoxic substances are excreted from both faces of leaves of barley and pea treated with these chemicals. Leaves from carboxin-treated plants, however, did not inhibit sensitive test organisms (S. roseus, L. scottii and Ustilago hordei), so their inhibitory action in vivo is deemed to result from hormonal imbalance in treated plants: carboxin-treated barley plants were greener than control plants, no guttation occurred on leaves, and stomata tended to remain open; treated barley and pea were more sensitive to drought than were controls. Carboxin-treated barley and pea plants sprayed with the cytokinin antagonist, abscisic acid, behaved as did plants without carboxin. It is considered likely that carboxin affects the epiphytes by creating a leaf surface incapable of sustaining them, possibly through dryness.     

Microplate Technique for Determining Accumulation of Metals by Algae

A microplate technique was developed to determine the conditions under which pure cultures of algae removed heavy metals from aqueous solutions. Variables investigated included algal species and strain, culture age (11 and 44 days), metal (mercury, lead, cadmium, and zinc), pH, effects of different buffer solutions, and time of exposure. Plastic, U-bottomed microtiter plates were used in conjunction with heavy metal radionuclides to determine concentration factors for metal-alga combinations. The technique developed was rapid, statistically reliable, and economical of materials and cells. Results (expressed as concentration factors) were in reasonably good agreement with literature values. All species of algae studied removed mercury from solution. Green algae proved better at accumulating cadmium than did blue-green algae. No alga studied removed zinc, perhaps because cells were maintained in the dark during the labeling period. Chlamydomonas sp. proved superior in ability to remove lead from solution.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Expression of Aureobasidium pullulans xynA in, and secretion of the xylanase from, Saccharomyces cerevisiae.

A previous report dealt with the cloning in Escherichia coli and sequencing of both the cDNA and genomic DNA encoding a highly active xylanase (XynA) of Aureobasidium pullulans (X.-L. Li and L. G. Ljungdahl, Appl. Environ. Microbiol. 60:3160-3166, 1994). Now we show that the gene was expressed in Saccharomyces cerevisiae under the GAL1 promoter in pYES2 and that its product was secreted into the culture medium. S. cerevisiae clone pCE4 with the whole open reading frame of xynA, including the part coding for the signal peptide, had xylanase activity levels of 6.7 U ml-1 in the cell-associated fraction and 26.2 U ml-1 in the culture medium 4 h after galactose induction. Two protein bands with sizes of 25 and 27 kDa and N-terminal amino acid sequences identical to that of APX-II accounted for 82% of the total proteins in the culture medium of pCE4. These proteins were recognized by anti-APX-II antibody. The results suggest that the XynA signal peptide supported the posttranslational processing of xynA product and the efficient secretion of the active xylanase from S. cerevisiae. Clones pCE3 and pGE3 with inserts of cDNA and genomic DNA, respectively, containing only the mature enzyme region attached by a Met codon had low levels of xylanase activity in the cell-associated fractions (1.6 U ml-1) but no activity in the culture media. No xylanase activity was detected in clone pGE4, which was the same as pCE4, except that pGE4 had a 59-bp intron in the signal peptide region. A comparison of the A. pullulans and S. cerevisiae signal peptides demonstrated that the XynA signal peptide was at least three times more efficient than those of S. cerevisiae invertase or mating alpha-factor pheromone in secreting the heterologous xylanase from S. cerevisiae cells.     

Susceptibility to Heavy Metals and Cadmium Accumulation in Aerobic and Anaerobic Thermophilic Microorganisms Isolated from Deep-Sea Hydrothermal Vents

Thirty thermophilic strains isolated from heavy metal-rich hydrothermal vent sites at Lau Basin were tested for their susceptibility to cadmium, zinc, cobalt, and nickel. The 14 aerobic spore formers belonging to the genus Bacillus, 6 anaerobic fermenters from the order Thermotogales, and 10 anaerobic sulfur reducers from the order Thermococcales could be clearly distinguished according to their metal susceptibilities. The Thermococcales were found to exhibit the highest resistance to cadmium and zinc, whereas Thermotogales were highly sensitive to these metals. In contrast, the Thermotogales displayed the highest resistance to cobalt ions. No clear distinction could be established between the metal susceptibilities of these strains and seven reference organisms used for comparative studies. Cadmium resistance, slightly inducible in some cadmium-resistant bacilli, was not plasmid mediated. The amount of cadmium immobilized by the Thermotogales was related to their level of resistance to this metal. Received: 24 February 2000 / Accepted: 28 March 2000     

Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation.

The degree of plasma membrane fatty acid unsaturation and the copper sensitivity of Saccharomyces cerevisiae are closely correlated. Our objective was to determine whether these effects could be accounted for by differential metal induction of lipid peroxidation. S. cerevisiae S150-2B was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Potassium efflux and colony count data indicated that sensitivity to both copper (redox active) and cadmium (redox inactive) was increased in 18:2-supplemented cells and particularly in 18:3-supplemented cells. Copper- and cadmium-induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements with the membrane probe trimethylammonium diphenylhexatriene. Levels of thiobarbituric acid-reactive substances (lipid peroxidation products) were up to twofold higher in 18:2-supplemented cells than in unsupplemented cells following metal addition, although this difference was reduced with prolonged incubation up to 3 h. Conjugated-diene levels in metal-exposed cells also increased with both the concentration of copper or cadmium and the degree of cellular fatty acid unsaturation; maximal levels were evident in 18:3-supplemented cells. The results demonstrate heavy metal-induced lipid peroxidation in a microorganism for the first time and indicate that the metal sensitivity of PUFA-enriched S. cerevisiae may be attributable to elevated levels of lipid peroxidation in these cells.     

Toxicity and mutual interactions of cadmium and zinc ions in normal and carcinogen-transformed mouse cells

The toxicity of chloride salts of physiological (zinc, manganese, nickel) and non physiological (cadmium) bivalent metal ions was studied in normal or carcinogen-transformed mouse embryo fibroblast cells. The dose response curves for toxicity to both types of cells exhibited similar shapes. The transformed cells, however, were about twice as sensitive to zinc toxicity as normal cells. When normal and transformed cells were grown together and incubated for several hours with an appropriate concentration of zinc, the malignant cells were selectively killed. Cadmium was much more toxic than the three other metal ions in both types of cells. Its toxic effect was reversed by simultaneous addition of zinc at nontoxic concentrations.Abbrevications CFA colony forming ability - MCA 3-methylcholanthrene     

Effects of trace metals on growth of yellow perch (Perca flavescens) as measured by RNA-DNA ratios

Synopsis Relationships between sublethal concentrations of cadmium and zinc in natural water and metal uptake by and growth of fish were investigated. RNA-DNA ratios and weight gain were used to assess seasonal growth differences between yellow perch populations from contaminated and control sites. Whole-body concentrations of cadmium and zinc in young-of-the-year perch (Perca flavescens) were significantly different between sites. Measurable growth differences did occur and were significantly correlated with cadmium levels. Growth differences that were prominent during mid-summer were reduced by late summer. RNA-DNA ratios were sensitive indicators of fish growth.     

Proteomic responses of the coccolithophore Emiliania huxleyi to zinc limitation and trace metal substitution

Zinc concentrations in pelagic surface waters are within the range that limits growth in marine phytoplankton cultures. However, the influence of zinc on marine primary production and phytoplankton communities is not straightforward due to largely uncharacterized abilities for some phytoplankton to access zinc species that may not be universally bioavailable and substitute zinc with cobalt or cadmium. We used a quantitative proteomic approach to investigate these strategies and other responses to zinc limitation in the coccolithophore Emiliania huxleyi, a dominant species in low zinc waters. Zinc limitation resulted in the upregulation of metal transport proteins (ZIP, TroA-like) and COG0523 metallochaperones. Some proteins were uniquely sensitive to growth under replete zinc, substitution of zinc with cobalt, or enhancement of growth with cadmium, and may be useful as biomarkers of zinc stress or substitution in situ. Several proteins specifically upregulated under cobalt-supported or cadmium-enhanced growth appear to reflect stress responses, despite titration of these metals to optimal nutritive levels. Relief from zinc limitation by zinc or cadmium resulted in increased expression of a δ-carbonic anhydrase. Our inability to detect metal binding enzymes that are specifically induced under cobalt- or cadmium-supported growth suggests cambialism is important for zinc substitution in E. huxleyi.     

Studies on the toxicity of zinc sulphate and of cadmium sulphate to stone loach Noemacheilus barbatulus (L.) in hard water

The toxicity of zinc sulphate and cadmium sulphate to stone loach Noemacheilus barbatulus was examined in an attempt to explain the distribution of the species in two polluted streams. Stone loach in hard water were more sensitive than rainbow trout to zinc, but much more resistant to cadmium. Their disappearance from the Willow Brook, Northants, in 1971–72 could be accounted for by zinc toxicity. A behavioural change, the loss of the instinct to hide during daylight, occurred in subsequently lethal concentrations of cadmium. The accumulation of cadmium in several tissues was also determined.     

Uptake of six heavy metals by oat as influenced by soil type and additions of cadmium,lead, zinc and copper

Summary The influence of heavy metal additions on availability and uptake of cadmium, lead, zinc, copper, manganese and iron by oat was studied. The experiments were carried out as pot experiments using sandy loam, sandy soil and organic soil. Selective extractants were used to remove metals held in different soil fractions.Lead and copper were preferently bound by organics and oxides, zinc by oxides and inorganics, and cadmium by inorganics and organics.Addition of cadmium to the soils resulted in higher cadmium concentrations in all plant parts but lower concentrations of lead, zinc, copper, manganese and iron, and the accumulation indexes of these metals were also lower when cadmium was added to the soil.Addition of cadmium plus lead, zinc and copper resulted in higher cadmium concentrations in leaves and straw of plants grown in sandy loam and sandy soil, but lower concentrations when plants were grown in organic soil as compared with the results when cadmium was added separately. The transfer of cadmium, lead, zinc and copper from soil to plant was greatest from sandy soil, and zinc and cadmium were more mobile in the plant than were lead and copper.Cadmium concentrations in leaves correlated significantly with CaCl₂ and CH₃COOH extractions in sandy loam and sandy soil and with CH₃COOH extractions in organic soil.Generally, the total metal uptake was lowest from organic soil.