Cristina A. Viegas

Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid

Plasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid [4-50 mg l-1 (0.03-0.35 mM), pH 4.0] was 1.5-fold higher than that in cells grown in its absence. The Km for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical. This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid. However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane. The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6.5 with octanoic acid at 100 mg l-1 or less (2.4 mg acid form plus 97.6 mg octanoate ion l-1). However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation. A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.

Enzymatic biotransformation of the azo dye Sudan Orange G with bacterial CotA-laccase

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30-80 degrees C) and more than 98% of Sudan Orange G is decolourised within 7h by using 1 U mL(-1) of CotA-laccase at 37 degrees C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

FLR1 gene (ORF YBR008c) is required for benomyl and methotrexate resistance in Saccharomyces cerevisiae and its benomyl‐induced expression is dependent on Pdr3 transcriptional regulator

In this work we report the disruption of a Saccharomyces cerevisiae ORF YBR008c (FLR1 gene) within the context of EUROFAN (EUROpean Functional Analysis Network) six‐pack programme, using a PCR‐mediated gene replacement protocol as well as the results of the basic phenotypic analysis of a deletant strain and the construction of a disruption cassette for inactivation of this gene in any yeast strain. We also show results extending the knowledge of the range of compounds to which FLR1 gene confers resistance to the antimitotic systemic benzimidazole fungicide benomyl and the antitumor agent methotrexate, reinforcing the concept that the FLR1 gene is a multidrug resistance (MDR) determinant. Our conclusions were based on the higher susceptibility to these compounds of flr1Δ compared with wild‐type and on the increased resistance of both flr1Δ and wild‐type strains upon increased expression of FLR1 gene from a centromeric plasmid clone. The present study also provides, for the first time, evidence that the adaptation of yeast cells to growth in the presence of benomyl involves the dramatic activation of FLR1 gene expression during benomyl‐induced latency (up to 400‐fold). Results obtained using a FLR1–lacZ fusion in a plasmid indicate that the activation of FLR1 expression in benomyl‐stressed cells is under the control of the transcriptional regulator Pdr3p. Indeed, PDR3 deletion severely reduces benomyl‐induced activation of FLR1 gene expression (by 85%), while the homologous Pdr1p transcription factor is apparently not involved in this activation. Copyright

Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae.

In this work, we report results on the functional analysis of Saccharomyces cerevisiae ORF YGR224w, predicted to code for an integral membrane protein, with 14 potential transmembrane segments, belonging to the major facilitator superfamily (MFS) of transporters which are required for multiple‐drug resistance (MDR). This MFS–MDR homologue is required for yeast adaptation to high stress imposed by low‐chain organic acids, in particular by acetic acid, and for resistance to azoles, especially to ketoconazole and fluconazole; the encoding gene was thus named the AZR1 gene. These conclusions were based on the higher susceptibility to these compounds of an azr1Δ deletion mutant strain compared with the wild‐type and on the increased resistance of both azr1Δ and wild‐type strains upon increased expression of the AZR1 gene from a centromeric plasmid clone. AZR1 gene expression reduces the duration of acetic acid‐induced latency, although the growth kinetics of adapted cells under acetic acid stress is apparently independent of AZR1 expression level. Fluorescence microscopy observation of the distribution of the Azr1–GFP fusion protein in yeast living cells indicated that Azr1 is a plasma membrane protein. Studies carried out to gain some understanding of how this plasma membrane putative transporter facilitates yeast adaptation to acetic acid did not implicate Azr1p in the alteration of acetic acid accumulation into the cell through the active efflux of acetate. Copyright

Evaluating a bioremediation tool for atrazine contaminated soils in open soil microcosms: The effectiveness of bioaugmentation and biostimulation approaches

A previously developed potential cleanup tool for atrazine contaminated soils was evaluated in larger open soil microcosms for optimization under more realistic conditions, using a natural crop soil spiked with an atrazine commercial formulation (Atrazerba FL). The doses used were 20x or 200x higher than the recommended dose (RD) for an agricultural application, mimicking over-use or spill situations. Pseudomonas sp. strain ADP was used for bioaugmentation (around 10(7) or 10(8) viable cells g(-1) of soil) and citrate for biostimulation (up to 4.8 mg g(-1) of soil). Bioremediation treatments providing fastest and higher atrazine biodegradation proved to differ according to the initial level of soil contamination. For 20x RD of Atrazerba FL, a unique inoculation with Pseudomonas sp. ADP (9 +/- 1 x 10(7) CFU g(-1)) resulted in rapid atrazine removal (99% of the initial 7.2 +/- 1.6 microg g(-1) after 8d), independent of citrate. For 200x RD, an inoculation with the atrazine-degrading bacteria (8.5 +/- 0.5 x 10(7) CFU g(-1)) supplemented with citrate amendment (2.4 mg g(-1)) resulted in improved biodegradation (87%) compared with bioaugmentation alone (79%), even though 7.8 +/- 2.1 microg of atrazine g(-1) still remained in the soil after 1 wk. However, the same amount of inoculum, distributed over three successive inoculations and combined with citrate, increased Pseudomonas sp. ADP survival and atrazine biodegradation (to 98%, in 1 wk). We suggest that this bioremediation tool may be valuable for efficient removal of atrazine from contaminated field soils thus minimizing atrazine and its chlorinated derivatives from reaching water compartments.

Effect of cinnamic acid on the growth and on plasma membrane H + -ATPase activity of Saccharomyces cerevisiae

Abstract Cinnamic acid and cinnamic acid derivatives occur in plants and fruits, providing a natural protection against infections by pathogenic microorganisms. They may also inhibit wine fermentation and other fruit juice fermentations by Saccharomyces cerevisiae and raise difficulties in the biological treatment of waste water from some food industries. In the present work, it is shown that cells of S. cerevisiae YPH499 grown at pH 4 and 30°C, in the presence of concentrations of cinnamic acid (20 or 35 mg/l) that reduce the maximum specific growth rate by 46 or 53%, respectively, exhibit a more active plasma membrane H + –ATPase than cells grown in its absence. This stimulatory effect was detected by assaying, during yeast growth in absence or presence of cinnamic acid, both the plasma membrane ATPase activity in crude membrane extracts and its action as a proton-pump by comparing extracellular acidification as a function of culture cell density. The lag-phase of approximately 8 h observed during cultivation in the presence of 20 mg/l cinnamic acid of yeast cells previously grown in its absence was eliminated by growing the inoculum in medium supplemented with the same concentration of cinnamic acid. These cinnamic acid adapted cells exhibited a more active plasma membrane H + –ATPase and this phenomenon may be due to and/or be among the mechanisms underlying the adaptative response to this toxic acid in yeast.

Toxicity of chlorinated phenoxyacetic acid herbicides in the experimental eukaryotic model Saccharomyces cerevisiae: role of pH and of growth phase and size of the yeast cell population

The inhibitory effect of the herbicides 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Saccharomyces cerevisiae growth is strongly dependent on medium pH (range 2.5-6.5). Consistent with the concept that the toxic form is the liposoluble undissociated form, at values close to their pK(a) (3.07 and 2.73, respectively) the toxicity is high, decreasing with the increase of external pH. In addition, the toxicity of identical concentrations of the undissociated acid form is pH independent, as observed with 2,4-dichlorophenol (2,4-DCP), an intermediate of 2,4-D degradation. Consequently, at pH values above 3.5 (approximately one unit higher than 2,4-D pK(a)), 2,4-DCP becomes more toxic than the original herbicide. A dose-dependent inhibition of growth kinetics and increased duration of growth latency is observed following sudden exposure of an unadapted yeast cell population to the presence of the herbicides. This contrasts with the effect of 2,4-DCP, which essentially affects growth kinetics. Experimental evidences suggest that the acid herbicides toxicity is not exclusively dependent on the liposolubility of the toxic form, as may essentially be the case of 2,4-DCP. An unadapted yeast cell population at the early stationary-phase of growth under nutrient limitation is significantly more resistant to short-term herbicide induced death than an exponential-phase population. Consequently, the duration of growth latency is reduced, as observed with the increase of the size of the herbicide stressed population. However, these physiological parameters have no significant effect either on growth kinetics, following growth resumption under herbicide stress, or on the growth curve of yeast cells previously adapted to the herbicides, indicating that their role is exerted at the level of cell adaptation.

Toxicity of octanoic acid in Saccharomyces cerevisiae at temperatures between 8.5 and 30°C

Octanoic acid causes the inhibition of growth, decrease of the biomass yield and yield of ATP, stimulation of the specific production of ethanol in Saccharomyces cerevisiae. The toxicity of this fatty acid is higher in cells grown at temperatures below 25°C, particularly below 15.5°C, as indicated by the enhancement of the exponential constant of growth inhibition by octanoic acid with the decrease of temperature. Moreover, its presence leads to an increase of the minimum temperature for growth. The intracellular pH (pHi) of cells harvested in the mid-exponential phase of growth at temperatures within the range 8.5–30°C decreased sharply from 6.8 – 6.9 down to 5.8 – 6.2 when octanoic acid is present at concentrations of the undissociated toxic form below 14 mg 1−1. However, with more inhibitory concentrations (up to 44.5 mg 1−1) the pHi only attained 5.7 – 6.1. This may result from the decreased synthesis of succinic and acetic acids in octanoic-acid-stressed cells and from the increased buffering capacity of their cytoplasm due to the reduced internal volume. The effect of octanoic acid on intracellular acidification is nearly independent of growth temperature within the range 8.5–30°C and cannot explain the augmentation of toxicity at low temperatures.

Comparison of two screening bioassays, based on the frog sciatic nerve and yeast cells, for the assessment of herbicide toxicity

Two different test systems, one based on the isolated sciatic nerve of an amphibian and the other on a microbial eukaryote, were used for the assessment of herbicide toxicity. More specifically, we determined the deleterious effects of increasing concentrations of herbicides of different chemical classes (phenoxyacetic acids, triazines, and acetamides), and of 2,4-dichlorophenol (2,4-DCP), a degradation product of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), on electrophysiological parameters and the vitality of the axons of the isolated sciatic nerve of the frog (Rana ridibunda) and on the growth curve of the yeast Saccharomyces cerevisiae based on microtiter plate susceptibility assays. The no-observed-effect-concentration (NOEC), defined as the maximum concentration of the tested compound that has no effect on these biological parameters, was estimated. In spite of the different methodological approaches and biological systems compared, the NOEC values were identical and correlated with the lipophilicity of the tested compounds. The relative toxicity established here, 2,4-DCP > alachlor, metolachlor >> metribuzin > 2,4-D, 2-methyl-4-chlorophenoxyacetic acid (MCPA), correlates with the toxicity indexes reported in the literature for freshwater organisms. Based on these results, we suggest that the relatively simple, rapid, and low-cost test systems examined here may be of interest as alternative or complementary tests for toxicological assessment of herbicides.

Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye-containing wastewaters.

The azoreductase PpAzoR from Pseudomonas putida shows a broader specificity for decolourization of azo dyes than CotA-laccase from Bacillus subtilis. However, the final products of PpAzoR activity exhibited in most cases a 2 to 3-fold higher toxicity than intact dyes themselves. We show that addition of CotA-laccase to PpAzoR reaction mixtures lead to a significant drop in the final toxicity. A sequential enzymatic process was validated through the use of 18 representative azo dyes and three model wastewaters that mimic real dye-containing effluents. A heterologous Escherichia coli strain was successfully constructed co-expressing the genes coding for both PpAzoR and CotA. Whole-cell assays of recombinant strain for the treatment of model dye wastewater resulted in decolourization levels above 80% and detoxification levels up to 50%. The high attributes of this strain, make it a promising candidate for the biological treatment of industrial dye containing effluents.