Modulation of chaperone gene expression in mutagenized Saccharomyces cerevisiae strains developed for recombinant human albumin production results in increased production of multiple heterologous proteins.

The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.

Antioxidant functions required for insusceptibility of Saccharomyces cerevisiae to tetracycline antibiotics.

Cu,Zn superoxide dismutase (Sod1) is required for insusceptibility of Saccharomyces cerevisiae to oxytetracycline (OTC). Here we report that Sod1 is also required for insusceptibility to doxycycline (DOX). Furthermore, among a range of antioxidant and redox balance mutants, mac1 and ctr1 deletion strains exhibited marked sensitization to OTC and DOX. Certain mutants exhibited a slight sensitivity to methacycline and minocycline. Addition of copper suppressed antibiotic sensitivity. Thus, intracellular copper as well as superoxide dismutase can be critical for eukaryotic tolerance of several tetracycline antibiotics.

Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases.

The GPX1, GPX2, and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs). We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes. Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs. PHGPx-like activity was detectable in extracts from wild-type S. cerevisiae and was diminished in extracts from gpx1 Delta, gpx2 Delta, and gpx3 Delta deletion mutants; PHGPx activity was almost absent in a gpx1 Delta/gpx2 Delta/gpx3 Delta triple mutant. Studies with cloned GPX1, GPX2, and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity. An S. cerevisiae gpx1 Delta/gpx2 Delta/gpx3 Delta mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3). This sensitivity to 18:3 was more marked than sensitivity to H(2)O(2). Unlike H(2)O(2) toxicity, delayed toxicity of 18:3 toward gpx1 Delta/gpx2 Delta/gpx3 Delta cells was correlated with the gradual incorporation of 18:3 into S. cerevisiae membrane lipids and was suppressible with alpha-tocopherol, an inhibitor of lipid peroxidation. The results show that the GPX genes of S. cerevisiae, previously reported to encode GPxs, encode PHGPxs (PHGPx1, PHGPx2, and PHGPx3) and that these enzymes protect yeast against phospholipid hydroperoxides as well as nonphospholipid peroxides during oxidative stress. This is the first report of an organism that expresses PHGPx from more than one gene and produces PHGPx in the absence of a GPx.

Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry.

Green fluorescent protein (GFP) has many advantages as a reporter molecule, but its stability makes it unsuitable for monitoring dynamic changes in gene expression, among other applications. Destabilized GFPs have been developed for bacterial and mammalian systems to counter this problem. Here, we extend such advances to the yeast model. We fused the PEST-rich 178 carboxyl-terminal residues of the G(1) cyclin Cln2 to the C terminus of yEGFP3 (a yeast- and FACS-optimized GFP variant), creating yEGFP3-Cln2(PEST). We tested the hybrid protein after integrating modules harbouring the yEGFP3 or yEGFP3-CLN2(PEST) ORFs into the Saccharomyces cerevisiae genome. yEGFP3- Cln2(PEST) had a markedly shorter half-life (t(1/2)) than yEGFP3; inhibition of protein synthesis with cycloheximide lead to a rapid decline in GFP content and fluorescence (t(1/2) approximately 30 min) in cells expressing yEGFP3-Cln2(PEST), whereas these parameters were quite stable in yEGFP3-expressing cells (t(1/2) approximately 7 h). We placed yEGFP3-CLN2(PEST) under the control of the CUP1 promoter, which is induced only transiently by copper. This transience was readily discernible with yEGFP3-Cln2(PEST), whereas yEGFP3 reported only on CUP1 switch-on, albeit more slowly than yEGFP3-Cln2(PEST). Cell cycle-regulated transcriptional activation/inactivation of the CLN2 promoter was also discernible with yEGFP3- Cln2(PEST), using cultures that were previously synchronized with nocodazole. In comparison to CLN2, expression from the ACT1 promoter was stable after release from nocodazole. We also applied a novel flow-cytometric technique for cell cycle analysis with asynchronous cultures. The marked periodicities of CLN2 and CLB2 (mitotic cyclin) transcription were readily evident from cellular yEGFP3-Cln2(PEST) levels with this non-perturbing approach. The results represent the first reported successful destabilization of a yeast-GFP. This new construct expands the range of GFP applications open to yeast workers.

Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp.

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10(5) to 10(6)/ml) at 22 degrees C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.

Copper/zinc-Superoxide dismutase is required for oxytetracycline resistance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S. cerevisiae hypersensitive to oxytetracycline (OTC): a sod1Delta mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 microg ml(-1), whereas concentrations of up to 1,000 microg ml(-1) had no effect on the growth of the wild type. OTC resistance was restored in the sod1Delta mutant by complementation with wild-type SOD1. The effect of OTC appeared to be cytotoxic and was not evident in a ctt1Delta (cytosolic catalase) mutant or in the presence of tetracycline. SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and sod1Delta strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1Delta mutant, but not the wild type, to OTC. We propose that Sod1p protects S. cerevisiae against a mode of OTC action that is dependent on oxidative damage.

Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae.

The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained. Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid non-perturbing approach based on flow cytometry. By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage. Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage. Furthermore, cells sorted by their Cu-sensitivity and-resistance and subsequently analyzed for DNA content were found predominantly to occupy G1/S and G2/M cell cycle stages, respectively. The oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G2/M phase S. cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells. The results indicate that differential Cu-sensitivity in a S. cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation.

Stimulation of strontium accumulation in linoleate-enriched Saccharomyces cerevisiae is a result of reduced Sr2+ efflux.

The influence of modified plasma membrane fatty acid composition on cellular strontium accumulation in Saccharomyces cerevisiae was investigated. Growth of S. cerevisiae in the presence of 1 mM linoleate (18:2) (which results in 18:2 incorporation to approximately 70% of total cellular and plasma membrane fatty acids, with no effect on growth rate) yielded cells that accumulated Sr2+ intracellularly at approximately twice the rate of S. cerevisiae grown without a fatty acid supplement. This effect was evident over a wide range of external Sr2+ concentrations (25 microM to 5 mM) and increased with the extent of cellular 18:2 incorporation. Stimulation of Sr2+ accumulation was not evident following enrichment of S. cerevisiae with either palmitoleate (16:1), linolenate (18:3) (n-3 and n-6 isomers), or eicosadienoate (20:2) (n-6 and n-9 isomers). Competition experiments revealed that Ca2+- and Mg2+-induced inhibition of Sr2+ accumulation did not differ between unsupplemented and 18:2-supplemented cells. Treatment with trifluoperazine (TFP) (which can act as a calmodulin antagonist and Ca2+-ATPase inhibitor), at a low concentration that precluded nonspecific K+ efflux, increased intracellular Sr2+ accumulation by approximately 3.6- and 1.4-fold in unsupplemented and 18:2-supplemented cells, respectively. Thus, TFP abolished the enhanced Sr2+ accumulation ability of 18:2-supplemented cells. Moreover, the rate of Sr2+ release from Sr2+-loaded fatty acid-unsupplemented cells was found to be at least twice as great as that from Sr2+-loaded 18:2-enriched cells. The influence of enrichment with other fatty acids on Sr2+ efflux was variable. The results reveal an enhanced Sr2+ accumulation ability of S. cerevisiae following 18:2-enrichment, which is attributed to diminished Sr2+ efflux activity in these cells.

Enrichment with a polyunsaturated fatty acid enhances the survival of Saccharomyces cerevisiae in the presence of tributyltin.

The toxicity of inorganic metal species towards Saccharomyces cerevisiae has been shown to be markedly dependent on cellular fatty acid composition. In this investigation, the influence of fatty acid supplementation on the toxicity of the lipophilic organometal, tributyltin was investigated. Growth of S. cerevisiae was increasingly inhibited when the tributyltin concentration was increased from 0 to 10 microM. However, the inhibitory effect was partly alleviated by supplementation of the medium with 1 mM linoleate (18:2), a treatment that leads to large-scale incorporation of this polyunsaturated fatty acid (to > 60% of total fatty acids) in yeast membrane lipids. Cells that were previously enriched with 18:2 also showed reduced loss of vitality compared to cells grown in the absence of a fatty acid supplement, when exposed to tributyltin. For example, addition of tributyltin to a concentration of 0.1 microM was associated with an approximate 10% reduction in the H+ efflux activity of 18:2-enriched cells, but a 70% reduction in that of fatty acid-unsupplemented cells. Despite the increased tributyltin resistance of 18:2-enriched S. cerevisiae, the level of cell-associated tributyltin was found to be approximately two-fold higher in these organisms than in fatty acid-unsupplemented cells. These results demonstrate an increased resistance of 18:2-enriched membranes to the direct toxic action(s) of tributyltin. This is in contrast to the previously reported effect of 18:2 enrichment on sensitivity of S. cerevisiae to inorganic metal cations.

Manganese toxicity towards Saccharomyces cerevisiae: dependence on intracellular and extracellular magnesium concentrations.

Inhibition of the growth of Saccharomyces cerevisiae was evident at concentrations of 0.5 mM Mn2+ or higher, but a tolerance to lower Mn2+ concentrations was observed. The inhibitory effects of 2.0 mM Mn2+ were eliminated by supplementing the medium with excess Mg2+ (10 mM), whereas addition of excess Ca2+ and K+ had negligible effect on Mn2+ toxicity. Growth inhibition by Mn2+, in the absence of a Mg2+ supplement, was attributed to Mn2+ accumulation to toxic intracellular levels. Mn levels in S. cerevisiae grown in Mg(2+)-supplemented medium were severalfold lower than those of cells growing in unsupplemented medium. Mn2+ toxicity was also influenced by intracellular Mg, as Mn2+ toxicity was found to be more closely correlated with the cellular Mg:Mn ratio than with cellular Mn levels alone. Cells with low intracellular levels of Mg were more susceptible to Mn2+ toxicity than cells with high cellular Mg, even when sequestered Mn2+ levels were similar. A critical Mg:Mn ratio of 2.0 was identified below which Mn2+ toxicity became acute. The results demonstrate the importance of intracellular and extracellular competitive interactions in determining the toxicity of Mn2+.

Influence of altered plasma membrane fatty acid composition on cesium transport characteristics and toxicity in Saccharomyces cerevisiae.

The influence of altered plasma membrane fatty acid composition on cesium uptake and toxicity was investigated in Saccharomyces cerevisiae. Detailed kinetic studies revealed that both the Vmax and Km values for Cs+ transport increased (by approximately twofold in the latter case) when S. cerevisiae was grown in medium supplemented with the polyunsaturated fatty acid linoleate. In addition, Cs+ uptake by linoleate-enriched cells was considerably less sensitive to the competitive effects of other monovalent cations (K+, Rb+, and NH4+) than that by unsupplemented cells. Stimulation of Cs+ uptake in the presence of certain K+ and Rb+ concentrations was only evident in linoleate-enriched S. cerevisiae. At 100 mM CsCl, the initial rate of Cs+ uptake was greater in linoleate-supplemented cells than in unsupplemented cells and this was reflected in a more rapid displacement of cellular K+. However, little difference in net Cs+ accumulation between linoleate-supplemented and unsupplemented cells was evident during prolonged incubation in buffer or during growth. Thus, Cs+ toxicity was similar in linoleate-supplemented and unsupplemented cells. The results were consistent with the Cs+ (K+) transport mechanism adopting an altered conformational state in linoleate-enriched S. cerevisiae.

Relationship between cadmium sensitivity and degree of plasma membrane fatty acid unsaturation in Saccharomyces cerevisiae.

The sensitivity of Saccharomyces cerevisiae to the redox-active metal copper has recently been found to be influenced by cellular fatty acid composition. This study sought to investigate whether fatty acid composition affected plasma membrane permeabilisation and whole-cell toxicity induced by the redox-inactive metal cadmium. S. cerevisiae NCYC 1383 was enriched with the polyunsaturated fatty acids linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous fatty acids resulted in them comprising more than 65% of the total fatty acids in plasma membrane lipids. Inhibition of cell division in the presence of Cd(NO3)2 was accentuated by growth in the presence of a polyunsaturated fatty acid. Furthermore, susceptibility to Cd(2+)-induced plasma membrane permeabilisation increased with the degree of fatty acid unsaturation. Thus, during exposure to Cd2+, K+ efflux from 18:2- and 18:3-enriched cells was up to 2.5-fold or 3-fold greater, respectively than that from unsupplemented cells. In addition, reductions in cell viability during exposure to Cd2+ were most marked in polyunsaturated-fatty-acid-supplemented cells. At certain times, unsupplemented Cd(2+)-exposed cells displayed up to 7-fold greater viability than supplemented Cd(2+)-exposed cells. The study demonstrates that the toxicity of the redox-inactive metal Cd2+ towards S. cerevisiae becomes markedly amplified with increased cellular and plasma membrane fatty acid unsaturation.

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.

Effects of biocides on Acanthamoeba castellanii as measured by flow cytometry and plaque assay.

The lethal effects of various biocides, in particular two agents (chlorhexidine diacetate and polyhexamethylene biguanide) on cysts and trophozoites of Acanthamoeba castellanii have been measured by both plaque assay and flow cytometry (FCM). Minimum amoebicidal concentrations against trophozoites were generally the same by the two methods whereas minimum cysticidal concentrations were occasionally higher by FCM. Concentrations of biguanides required to produce an equal reduction in viability were also higher by FCM than by plaque assay.

Manganese uptake and toxicity in magnesium-supplemented and unsupplemented Saccharomyces cerevisiae.

The magnesium content of Saccharomyces cerevisiae was found to vary by up to fivefold at differing stages of batch growth and during growth in the presence of differing magnesium concentrations. Excess Mg was primarily sequestered in vacuoles. Mn(2+)-uptake experiments revealed that Mg-enriched cells had a markedly reduced capacity for Mn2+ accumulation. For example, after 6 h incubation in the presence of 50 microM Mn2+, Mn levels were approximately twofold higher in cells previously grown in unsupplemented medium than in those from Mg-supplemented medium. These differences were further accentuated at higher Mn2+ concentrations and were not attributable to altered cell-surface charge or altered cell-surface Mn2+ binding. Cellular Mg status also influenced Mn toxicity towards S. cerevisiae. During exposure to 5 mM Mn2+, 50% reductions in the viability of cells with initial Mg contents of approximately 1400 and 2700 nmol (10(9) cells)-1 occurred after approximately 1.6 h and 3.6 h respectively. In cells containing 3300 nmol Mg (10(9) cells)-1, more than 75% viability was still maintained after 7 h incubation with 5 mM Mn2+. It is concluded that Mn2+ uptake and toxicity in S. cerevisiae are strongly influenced by intracellular Mg, possibly through Mg-dependent regulation of divalent-cation transport activity.

Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition.

One major mechanism of copper toxicity towards microorganisms is disruption of plasma membrane integrity. In this study, the influence of plasma membrane fatty acid composition on the susceptibility of Saccharomyces cerevisiae to Cu2+ toxicity was investigated. Microbial fatty acid composition is highly variable, depending on both intrinsic and environmental factors. Manipulation was achieved in this study by growth in fatty acid-supplemented medium. Whereas cells grown under standard conditions contained only saturated and monounsaturated fatty acids, considerable incorporation of the diunsaturated fatty acid linoleate (18:2) (to more than 65% of the total fatty acids) was observed in both whole-cell homogenates and plasma membrane-enriched fractions from cells grown in linoleate-supplemented medium. Linoleate enrichment had no discernible effect on the growth of S. cerevisiae. However, linoleate-enriched cells were markedly more susceptible to copper-induced plasma membrane permeabilization. Thus, after addition of Cu(NO3)2, rates of cellular K+ release (loss of membrane integrity) were at least twofold higher from linoleate-supplemented cells than from unsupplemented cells; this difference increased with reductions in the Cu2+ concentration supplied. Levels of cellular Cu accumulation were also higher in linoleate-supplemented cells. These results were correlated with a very marked dependence of whole-cell Cu2+ toxicity on cellular fatty acid unsaturation. For example, within 10 min of exposure to 5 microM Cu2+, only 3% of linoleate-enriched cells remained viable (capable of colony formation). In contrast, 100% viability was maintained in cells previously grown in the absence of a fatty acid supplement. Cells displaying intermediate levels of linoleate incorporation showed intermediate Cu2+ sensitivity, while cells enriched with the triunsaturated fatty acid linolenate (18:3) were most sensitive to Cu2+. These results demonstrate for the first time that changes in cellular and plasma membrane fatty acid compositions can dramatically alter microbial sensitivity to copper.

Temperature-dependent changes in plasma-membrane lipid order and the phagocytotic activity of the amoeba Acanthamoeba castellanii are closely correlated.

The relationship between temperature-dependent changes in phagocytotic activity of Acanthamoeba castellanii and the fatty acid composition and physical properties of plasma membrane-enriched fractions were determined in cultures acclimated to 30 degrees C and 15 degrees C. Chilling (from 30 degrees C to 15 degrees C) had a very pronounced short-term inhibitory effect on phagocytosis only in stationary-phase cultures, which displayed a low degree of fatty acid unsaturation. A subsequent increase in phagocytosis by these cells was correlated with a low-temperature-induced increase in fatty acid unsaturation (shown previously [Jones, Lloyd and Harwood (1993) Biochem. J. 296, 183-188] to be due to n-6 desaturase induction). Plasma membrane-enriched fractions from 15 degrees C-acclimated cells also showed a marked increase in the relative proportion of polyunsaturated fatty acids. Steady-state fluorescence depolarization studies, using the membrane probe diphenylhexatriene, revealed increases in plasma membrane order with decreasing assay temperature. Over the upper assay-temperature range (25-40 degrees C), fluorescence anisotropy values were higher in membranes from 30 degrees C-acclimated cells; a 3.3 degrees C relative displacement of plots indicated that temperature-induced changes in membrane lipid composition compensated for approx. 22% of the ordering effect of low temperature. Changes in the temperature-dependence of fluorescence anisotropy, possibly corresponding to lateral phase separations or alterations in other bulk physical properties of membranes, occurred between 20 and 25 degrees C in membranes from 30 degrees C-acclimated cells and between 15 and 20 degrees C in membranes from 15 degrees C-acclimated cells. Fluorescence anisotropy plots were superimposed at assay temperatures between 5 and 15 degrees C. Short-term phagocytotic rates in whole cells decreased with assay temperature. Arrhenius discontinuities in rates of phagocytosis occurred at approx. 25.0 degrees C and 17.5 degrees C in 30 degrees C- and 15 degrees C-acclimated cells respectively, and in each case were thus within the temperature ranges of slope-change in the corresponding fluorescence anisotropy plots. The results show a direct correlation between plasma membrane fatty acid unsaturation, membrane physical properties and phagocytotic activity in A. castellanii. Therefore, a specific integrated physiological process has been correlated with fatty acid desaturase induction for the first time.