3D map distribution of metallic nanoparticles in whole cells using MeV ion microscopy.

In this work, a new tool was developed, the MORIA program that readily translates Rutherford backscattering spectrometry (RBS) output data into visual information, creating a display of the distribution of elements in a true three-dimensional (3D) environment. The program methodology is illustrated with the analysis of yeast Saccharomyces cerevisiae cells, exposed to copper oxide nanoparticles (CuO-NP) and HeLa cells in the presence of gold nanoparticles (Au-NP), using different beam species, energies and nuclear microscopy systems. Results demonstrate that for both cell types, the NP internalization can be clearly perceived. The 3D models of the distribution of CuO-NP in S. cerevisiae cells indicate the nonuniform distribution of NP in the cellular environment and a relevant confinement of CuO-NP to the cell wall. This suggests the impenetrability of certain cellular organelles or compartments for NP. By contrast, using a high-resolution ion beam system, discretized agglomerates of Au-NP were visualized inside the HeLa cell. This is consistent with the mechanism of entry of these NPs in the cellular space by endocytosis enclosed in endosomal vesicles. This approach shows RBS to be a powerful imaging technique assigning to nuclear microscopy unparalleled potential to assess nanoparticle distribution inside the cellular volume.

A quantitative proteomic approach to highlight Phragmites sp. adaptation mechanisms to chemical stress induced by a textile dyeing pollutant.

Phragmites sp. is present worldwide in treatment wetlands though the mechanisms involved in the phytoremediation remain unclear. In this study a quantitative proteomic approach was used to study the prompt response and adaptation of Phragmites to the textile dyeing pollutant, Acid Orange 7 (AO7). Previously, it was demonstrated that AO7 could be successfully removed from wastewater and mineralized in a constructed wetland planted with Phragmites sp. This azo dye is readily taken up by roots and transported to the plant aerial part by the xylem. Phragmites leaf samples were collected from a pilot scale vertical flow constructed wetland after 0.25, 3.25 and 24.25h exposure to AO7 (400mgL-1) immediately after a watering cycle used as control. Leaf soluble protein extraction yielded an average of 1560 proteins in a broad pI range (pH3-10) by two-dimensional gel electrophoresis. A time course comparative analysis of leaf proteome revealed that 40 proteins had a differential abundance compared to control (p<0.05) within a 3.25h period. After 24.25h in contact with AO7, leaf proteome was similar to control. Adaptation to AO7 involved proteins related with cellular signalling (calreticulin, Ras-related protein Rab11D and 20S proteasome), energy production and conversion (adenosine triphosphate synthase beta subunit) carbohydrate transport and metabolism (phosphoglucose isomerase, fructose-bisphosphate aldolase, monodehydroascorbate reductase, frutockinase-1 and Hypothetical protein POPTR_0003s12000g and the Uncharacterized protein LOC100272772) and photosynthesis (sedoheptulose-1,7-bisphosphatase and ferredoxin-NADP+ reductase). Therefore, the quantitative proteomic approach used in this work indicates that mechanisms associated with stress cell signalling, energy production, carbohydrate transport and metabolism as well as proteins related with photosynthesis are key players in the initial chemical stress response in the phytoremediation process of AO7.

Investigation of the multifaceted iron acquisition strategies of Burkholderia cenocepacia.

Burkholderia cenocepacia is a bacterial pathogen which causes severe respiratory infections in cystic fibrosis (CF). These studies were aimed at gaining an insight into the iron acquisition strategies of B. cenocepacia. In iron restricted conditions, genes associated with the synthesis and utilisation of ornibactin (pvdA, orbA, orb F) were significantly upregulated compared to the expression of pyochelin associated genes (pchD, fptA). In the absence of alternative iron sources, B. cenocepacia J2315 and 715j utilised ferritin and haemin, but not transferrin or lactoferrin for growth. Significantly, mutants unable to produce ornibactin, (715j-orbI) or ornibactin and pyochelin, (715j-pobA), utilised haemin and ferritin more efficiently than the wild-type. Moreover, both mutants were also able to utilise lactoferrin for growth (P ≤ 0.01) and additionally 715j-pobA utilised transferrin (P ≤ 0.01), potentially facilitating adaptation to the host environment. Furthermore, B. cenocepacia increased ornibactin gene expression in response to pyoverdine from Pseudomonas aeruginosa (P ≤ 0.01), demonstrating the capacity to compete for iron in co-colonised niches. Pyoverdine also significantly diminished the growth of B. cenocepacia (P < 0.001) which was related to its iron chelating activity. In a study of three B. cenocepacia sequential clonal isolates obtained from a CF patient over a 3.5 year period, ornibactin upregulation in response to pyoverdine was less pronounced in the last isolate compared to the earlier isolates, as was growth in the presence of haemin and ferritin, indicating alternative iron acquisition mechanism(s) may dominate as chronic infection progresses. These data demonstrate the multifaceted iron acquisition strategies of B. cenocepacia and their capacity to be differentially activated in the presence of P. aeruginosa and during chronic infection.

The Pht1;9 and Pht1;8 transporters mediate inorganic phosphate acquisition by the Arabidopsis thaliana root during phosphorus starvation.

• The activation of high-affinity root transport systems is the best-conserved strategy employed by plants to cope with low inorganic phosphate (Pi) availability, a role traditionally assigned to Pi transporters of the Pht1 family, whose respective contributions to Pi acquisition remain unclear. • To characterize the Arabidopsis thaliana Pht1;9 transporter, we combined heterologous functional expression in yeast with expression/subcellular localization studies and reverse genetics approaches in planta. Double Pht1;9/Pht1;8 silencing lines were also generated to gain insight into the role of the closest Pht1;9 homolog. • Pht1;9 encodes a functional plasma membrane-localized transporter that mediates high-affinity Pi/H⁺ symport activity in yeast and is highly induced in Pi-starved Arabidopsis roots. Null pht1;9 alleles exhibit exacerbated responses to prolonged Pi limitation and enhanced tolerance to arsenate exposure, whereas Pht1;9 overexpression induces the opposite phenotypes. Strikingly, Pht1;9/Pht1;8 silencing lines display more pronounced defects than the pht1;9 mutants. • Pi and arsenic plant content analyses confirmed a role of Pht1;9 in Pi acquisition during Pi starvation and arsenate uptake at the root-soil interface. Although not affecting plant internal Pi repartition, Pht1;9 activity influences the overall Arabidopsis Pi status. Finally, our results indicate that both the Pht1;9 and Pht1;8 transporters function in sustaining plant Pi supply on environmental Pi depletion.

Qualitative modelling and formal verification of the FLR1 gene mancozeb response in Saccharomyces cerevisiae.

BACKGROUND: Qualitative models allow understanding the relation between the structure and the dynamics of gene regulatory networks. The dynamical properties of these models can be automatically analysed by means of formal verification methods, like model checking. This facilitates the model-validation process and the test of new hypotheses to reconcile model predictions with the experimental data. RESULTS: The authors report in this study the qualitative modelling and simulation of the transcriptional regulatory network controlling the response of the model eukaryote Saccharomyces cerevisiae to the agricultural fungicide mancozeb. The model allowed the analysis of the regulation level and activity of the components of the gene mancozeb-induced network controlling the transcriptional activation of the FLR1 gene, which is proposed to confer multidrug resistance through its putative role as a drug eflux pump. Formal verification analysis of the network allowed us to confront model predictions with the experimental data and to assess the model robustness to parameter ordering and gene deletion. CONCLUSIONS: This analysis enabled us to better understand the mechanisms regulating the FLR1 gene mancozeb response and confirmed the need of a new transcription factor for the full transcriptional activation of YAP1. The result is a computable model of the FLR1 gene response to mancozeb, permitting a quick and cost-effective test of hypotheses prior to experimental validation.

Refining current knowledge on the yeast FLR1 regulatory network by combined experimental and computational approaches.

Multidrug resistance is often the result of the activation of drug efflux pumps able to catalyze the extrusion of the toxic compound to the outer medium, this activation being frequently controlled at the transcriptional level. Transcriptional regulation in the model eukaryote S. cerevisiae is the result of the interaction and cross-talk between networks of transcription factors. This is the case of the transcriptional activation of the FLR1 gene occurring in response to stress induced by the agricultural fungicide mancozeb in yeast. FLR1 up-regulation depends on the integrated action of Yap1, a key regulator of oxidative stress response, Pdr3 and Yrr1, two of the transcription factors controlling multidrug resistance, and Rpn4, a regulator of proteasome gene expression, which interplay to produce the observed transcriptional up-shift. Based on the expression profiles of FLR1, YAP1, PDR3, YRR1 and RPN4 registered during yeast adaptation to stress induced by mancozeb and using a qualitative modeling approach, a model of the FLR1 regulatory network was built, and the response of S. cerevisiae to mancozeb stress was simulated. The use of a qualitative approach is especially useful to overcome the lack of enough quantitative data on kinetic parameters and molecular concentrations, permitting the immediate focus on the qualitative behavior of the system. This Systems Biology approach allowed the identification of essential features of the early yeast response to fungicide stress. The resulting model allowed the formulation of new hypotheses, in a quick and cost effective manner, on the qualitative behavior of the system following mancozeb challenge, some of which were validated experimentally. In particular, Pdr3 and Yrr1 were shown to directly control FLR1 up-regulation in mancozeb-challenged cells, based on the analysis of the effect of the inactivation of their putative binding sites in the FLR1 promoter. Furthermore, the inter-dependent role of Yap1 and Yrr1 in the regulation of PDR3 and RPN4 was brought to light, this joint activity possibly being extensible to eight other genes involved in multidrug resistance. The FLR1 network structure was revised, based on the comparison between simulated and experimental gene expression data in the double deletion mutant strains Δyrr1Δpdr3 and Δyrr1Δrpn4, and an additional, still unidentified, transcription factor was found to be required to fully explain the behavior of the network.

Variation of the antimicrobial susceptibility profiles of Burkholderia cepacia complex clonal isolates obtained from chronically infected cystic fibrosis patients: a five-year survey in the major Portuguese treatment center.

The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia complex (Bcc) bacteria requires extensive and aggressive antibiotics therapy, exposing these bacteria to prolonged antibiotics-selective pressure. In the present study, we have compared the susceptibility patterns to 13 antimicrobials of 94 Bcc isolates obtained from 15 Portuguese CF patients in the course of chronic infection during a five-year survey. These isolates were previously genotyped and represent 11 different strains of the species B. cenocepacia (subgroups A and B), B. cepacia, B. multivorans, and B. stabilis. The results are consistent with the notion that CF Bcc isolates are resistant to the most clinically relevant antimicrobials and suggest an uneven distribution of resistance rates among the different species, with B. cenocepacia subgroup A isolates being the most resistant. Phenotypic variants exhibiting differences in the antimicrobial susceptibility patterns were obtained from the sputum samples of clinically deteriorated CF patients during chronic lung infection. The isolation of resistant variants coincided with periods of pulmonary exacerbation and antibiotics therapy.

Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.

The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P2(1) crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P2(1) has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, beta = 105.2 degrees . Model building and refinement are currently under way.

Saccharomyces cerevisiae adaptation to weak acids involves the transcription factor Haa1p and Haa1p-regulated genes.

The understanding of the molecular mechanisms that may contribute to counteract the deleterious effects of organic acids as fungistatic agents is essential to guide suitable preservation strategies. In this work, we show that the recently identified transcription factor Haa1p is required for a more rapid adaptation of a yeast cell population to several weak acid food preservatives. Maximal protection is exerted against the short-chain length acetic or propionic acids. The transcription of nine Haa1p-target genes, many of which are predicted to encode membrane proteins of unknown or poorly characterized function, is activated under weak acid stress. The Haa1-regulated genes required for a more rapid yeast adaptation to weak acids include TPO2 and TPO3, encoding two predicted plasma membrane multidrug transporters of the major facilitator superfamily, and YGP1, encoding a poorly characterized cell wall glycoprotein. The acetic acid-induced prolongation of the lag phase of unadapted cell populations lacking HAA1 or TPO3, compared with wild-type population, was correlated with the level of the acid accumulated into the stressed cells.

Adaptative responses in yeast to the herbicide 2-methyl-4-chlorophenoxyacetic acid at the level of intracellular pH homeostasis.

AIMS: The objective of this work was to examine adaptative responses occurring in Saccharomyces cerevisiae following exposure to the herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA). METHODS AND RESULTS: The exposure of a yeast cell population to MCPA concentrations of moderate toxicity led to a period of latency before eventual resumption of inhibited growth. During this period of adaptation, the plasma membrane (PM) H+-ATPase was activated, in coordination with the decrease of intracellular pH (pHi), cell viability and average cell volume. The in vivo activation of this ATPase was demonstrated either by assaying PM-ATPase activity in membrane suspensions extracted from cells grown in the presence or absence of MCPA or by measuring the in vivo H+-pumping activity in the same cells. The PM-H+-ATPase activation could not be attributed to transcriptional activation of the encoding genes PMA1 and PMA2. CONCLUSIONS: The activity of PM-H+-ATPase was stimulated and the internal cell volume decreased during yeast adaptation to growth under MCPA stress. Based on the values estimated for the pHi, we hypothesize that these cell responses may contribute to the restoration of pHi homeostasis during recovery from MCPA stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is a contribution to the understanding of the toxic effects of the herbicide MCPA and of physiological mechanisms underlying adaptation to MCPA, in the eukaryotic model S. cerevisiae. Results may be useful to elucidate the adaptation mechanisms to this xenobiotic compound in more complex and experimentally less-accessible eukaryotes. They also provide indications to assist the use of yeast cells as a bioassay system to assess the toxicity of phenoxyacetic acid herbicides and of other lipophilic xenobiotics, aiming at reducing the use of animals in toxicity testing.

Activation and significance of vacuolar H+-ATPase in Saccharomyces cerevisiae adaptation and resistance to the herbicide 2,4-dichlorophenoxyacetic acid.

The stimulation of the activity of the H(+)-ATPase present in the vacuolar membrane (V-ATPase) of Saccharomyces cerevisiae is here described in response to a moderate stress induced by 2,4-dichlorophenoxyacetic acid (2,4-D). This in vivo activation (up to 5-fold) took place essentially during the adaptation period, preceding cell division under herbicide stress, in coordination with a marked activation of plasma membrane H(+)-ATPase (PM-ATPase) (up to 30-fold) and the decrease of intracellular and vacuolar pH values, suggesting that activation may be triggered by acidification. Single deletion of VMA1 and genes encoding other V-ATPase subunits led to a more extended period of adaptation and to slower growth under 2,4-D stress. Results suggest that a functional V-ATPase is required to counteract, more rapidly and efficiently, the dissipation of the physiological H(+)-gradient across vacuolar membrane registered during 2,4-D adaptation.

Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase.

The ces10 gene of the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461 was cloned and sequenced. Multi-sequence alignment of the deduced protein indicated that Ces10 belongs to the serine hydrolase family with a potential catalytic triad comprising Ser(153) (within the G-X-S-X-G consensus sequence), His(75) and Asp(125). The mixed block results obtained following pattern search and the low identities detected in a BLAST analysis indicate that Ces10 is significantly different from other characterised bacterial esterases/lipases. Nevertheless, the Ces10 amino acid sequence showed 45% similarity with Rhodococcus sp. heroin esterase and 48% with Bacillus subtilis p-nitrobenzyl esterase. Ces10, with a predicted molecular mass of 30,641 Da, was overproduced in Escherichia coli and purified to homogeneity in a histidine-tagged form. Enzyme assays using p-nitrophenyl-esters (p-NP-esters) with different acyl chain-lengths as the substrate confirmed the anticipated esterase activity. Ces10 exhibited a marked preference for short-chain fatty acids, yielding the highest activity with p-NP-propionate (optimal pH 7.4, optimal temperature 37 degrees C).

Characterization of the ugpG gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer Sphingomonas paucimobilis ATCC 31461.

The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g.

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.

Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes, enzymes and exopolysaccharide production engineering.

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production.

Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461.

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929-939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH(2) and -CO(2)H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH(2) (GelK(NH2)) and -CO(2)H (GelK(COOH)) halves were overexpressed in Escherichia coli. GelK and GelK(NH2) were present in both the soluble and membrane fractions of E. coli, whereas GelK(COOH) was only present in the soluble fraction. GelK catalysed the transfer of [(14)C]glucuronic acid from UDP-[(14)C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by beta-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PP(i)-lipid moiety is needed for enzymic activity. Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.

Mechanisms underlying the acquisition of resistance to octanoic-acid-induced-death following exposure of Saccharomyces cerevisiae to mild stress imposed by octanoic acid or ethanol.

Acquisition of resistance to lethal concentrations of octanoic acid was induced in cells of Saccharomyces cerevisiae grown in the presence of sublethal concentrations of this lipophilic acid or following rapid exposure (1 h) of unadapted yeast cells to mild stress imposed by the same acid. Experimental evidence indicated that the referred adaptation involved de novo protein synthesis, presumably due to the rapid induction of a plasma membrane transporter which mediates the active efflux of octanoate out of the cell. Rapid exposure of cells to mild ethanol stress also led to increased resistance to lethal concentrations of octanoic acid. This cross-resistance to octanoic-acid-induced death was below the level of resistance induced by mild octanoic acid stress and did not involve induction of the active expulsion of octanoate out of the cell. However, the rapid exposure of yeast cells to octanoic acid or ethanol led to the activation of plasma membrane H+-ATPase. The physiological role of the two stress responses examined during the present study, namely, the active efflux of octanoate specifically induced by octanoic acid and the stimulation of plasma membrane H+-ATPase activity, is discussed.

The activity of plasma membrane H(+)-ATPase is strongly stimulated during Saccharomyces cerevisiae adaptation to growth under high copper stress, accompanying intracellular acidification.

For the adaptation of cells of Saccharomyces cerevisiae, a period of latency is necessary before exponential growth is resumed in a medium supplemented with a highly inhibitory concentration of copper. In this work, we have examined some physiological responses occurring during this period of adaptation. The results revealed that plasma membrane H(+)-ATPase (PM-ATPase) activity is strongly stimulated (up to 24-fold) during copper-induced latency in growth medium with glucose, reaching maximal levels when the cells were about to start inhibited exponential growth. This in vivo activation of the ATPase activity by copper was accompanied by the stimulation of the H(+)-pumping activity of the enzyme in vivo and was essentially due to the increase of the apparent V(max) for MgATP. Although the exact molecular basis of the reported plasma membrane ATPase activation was not clarified, no increase in the mRNA levels from the encoding genes PMA1 and PMA2 was apparently detected during copper-induced latency. The physiological response reported here may allow the cells to cope with copper-induced lipid peroxidation and consequent decrease in plasma membrane lipid ordering and increase in the non-specific permeability to protons. The consequences of these copper deleterious effects were revealed by the decrease of the intracellular pH (pH(i)) of the yeast population, from approximately pH(i) 6 to pH(i) 5, during copper-induced latency in growth medium at pH 4.3. The time-dependent patterns of plasma membrane ATPase activation and of the decrease of pH(i) during the period of adaptation to growth with copper correlate, suggesting that the regulation of this membrane enzyme activity may be triggered by intracellular acidification. Consistent with this idea, when exponential growth under copper stress was resumed and the pH(i) of the yeast population recovered up to physiological values, plasma membrane ATPase activity simultaneously decreased from the highly stimulated level attained during the adaptation period of latency.

Resistance and adaptation to quinidine in Saccharomyces cerevisiae: role of QDR1 (YIL120w), encoding a plasma membrane transporter of the major facilitator superfamily required for multidrug resistance.

As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frame YIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1.

Transcriptional activation of FLR1 gene during Saccharomyces cerevisiae adaptation to growth with benomyl: role of Yap1p and Pdr3p.

The adaptation of Saccharomyces cerevisiae to growth in the presence of the antimitotic fungicide benomyl involves the dramatic activation of FLR1 transcription, taking place during benomyl-induced latency following sudden exposure to the fungicide. FLR1 gene encodes a plasma membrane transporter of the major facilitator superfamily (MFS) conferring resistance to multiple drugs, in particular to benomyl. FLR1 activation is completely abolished in a mutant devoided of YAP1 gene being exerted by Yap1p either directly or via Pdr3p. YAP1 gene was proved to be a determinant of benomyl resistance; the duration of the adaptation period preceding cell division under benomyl stress was longer for the Deltayap1 population, presumably due to the abolishment of FLR1 activation during latency. Although benomyl resistance mediated by Yap1p is reduced in a FLR1 deletion mutant, results also indicate that Yap1p may have other target genes that confer benomyl resistance in yeast.