Chemogenomic and transcriptome analysis identifies mode of action of the chemosensitizing agent CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine)
© Batova et al; licensee BioMed Central Ltd. 2010
Received: 23 October 2009
Accepted: 4 March 2010
Published: 4 March 2010
CTBT (7-chlorotetrazolo [5,1-c]benzo[1,2,4]triazine) increases efficacy of commonly used antifungal agents by an unknown mechanism. It increases the susceptibility of Saccharomyces cerevisiae, Candida albicans and Candida glabrata cells to cycloheximide, 5-fluorocytosine and azole antimycotic drugs. Here we elucidate CTBT mode of action with a combination of systematic genetic and transcriptome analysis.
To identify the cellular processes affected by CTBT, we screened the systematic haploid deletion mutant collection for CTBT sensitive mutants. We identified 169 hypersensitive deletion mutants. The deleted genes encode proteins mainly involved in mitochondrial functions, DNA repair, transcription and chromatin remodeling, and oxidative stress response. We found that the susceptibility of yeast cells to CTBT depends on molecular oxygen. Transcriptome analysis of the immediate early response to CTBT revealed rapid induction of oxidant and stress response defense genes. Many of these genes depend on the transcription factors Yap1 and Cin5. Yap1 accumulates rapidly in the nucleus in CTBT treated cells suggesting acute oxidative stress. Moreover, molecular calculations supported a superoxide generating activity of CTBT. Superoxide production in vivo by CTBT was found associated to mitochondria as indicated by oxidation of MitoSOX Red.
We conclude that CTBT causes intracellular superoxide production and oxidative stress in fungal cells and is thus enhancing antimycotic drug effects by a secondary stress.
Fugal pathogens pose a serious threat to immunocompromised persons. Despite many antifungal agents interfering with metabolism and growth of fungal cells a limited number of compounds are being used for treatment of mycotic diseases caused by human pathogenic fungal species. Over the past two decades, the number of invasive fungal infections has increased in the clinical setting. Candida sp. is the fourth most common pathogen identified, and other pathogens such as Cryptococcus sp., Aspergillus sp., and Fusarium sp., have a high morbidity and mortality. In addition, the incidence of mycoses caused by opportunistic fungi is rising . CTBT, 7-chlorotetrazolo [5,1-c]benzo[1,2,4]triazine, has antifungal activity and enhances the efficacy of other antifungals with different targets such as cycloheximide, fluconazole or 5-fluorocytosine . The molecular mechanism of CTBT action has not yet been resolved.
Currently used antifungals belong to three major classes of agents: azoles, polyenes, and echinocandines . These compounds target ergosterol biosynthesis, membrane functions and cell wall biosynthesis. Additionally, other currently applied compounds are the pyrimidine, 5-fluorocytosine (5-FC), which acts by inhibiting RNA and DNA synthesis  and ciclopiroxolamine which seems to induce oxidative stress and iron deprivation . The intrinsic resistance of human fungal pathogens to these substances is different. Echinocandins are effective in Candida prevention and offer a greater spectrum of activity across the various Candida species, including also C. krusei and C. glabrata, which are not reliably covered by azoles e.g. fluconazole .
Fungal drug resistance mechanisms involve decreased drug uptake, increased drug export, overexpression or structural modification of the drug target protein [7, 8]. Reversal of antifungal drug effectiveness in yeast cells mediated by efflux has been reported for a variety of substances targeting different molecular processes. These are for example the immunosuppressive agents FK506 and cyclosporine [9–13]. To overcome drug resistance of human fungal pathogens, new antifungals with novel cellular targets  and multidrug resistance reversal agents rendering drug resistant strains sensitive to commercially used antifungals are being developed [15, 16] but have not surfaced as yet. Studies evaluating combinations of antifungals have shown synergistic and additive activity. However, caution is required, because some antifungal combinations have demonstrated antagonistic activity. Controlled clinical trials are still necessary to explore the various efficacious antifungal combinations . Since the common antifungals are mainly targeting membrane and cell wall components, efficient combination therapy might be reached by involving substances with an alternative mode of action.
The site and mode of CTBT action have not yet been resolved. CTBT displayed a weak antifungal activity which was unaffected by deletion of the PDR1 and PDR3 genes encoding the main transcription activators involved in the control of multidrug resistance in Saccharomyces cerevisiae[18, 19]. Yeast cells grown in its presence had altered sterol composition and were more sensitive to this compound in the yap1Δ genetic background . Here we report insights gained for the mode of CTBT action by systematic identification of yeast genes required for resistance to CTBT in combination with transcriptome analysis. We will show that CTBT causes an unexpected dramatic response to oxidative stress including damage to mitochondria and genomic DNA. These results provide a model for CTBT action and indicate that its synergic effect with commonly used antifungal drugs is due to the combination of oxidative and other stresses.
CTBT action depends on molecular oxygen and is connected by mitochondrial functions
Identification of yeast deletion mutants with increased CTBT susceptibility
To gain more insight into CTBT action we systematically identified mutants with altered sensitivity. We screened the S. cerevisiae haploid deletion mutant collection for altered growth in the presence of CTBT. The wild type strain BY4741 from which the EUROSCARF collection has been derived was unable to grow on YPD medium supplemented with 6 μg/ml of CTBT. Therefore, the hypersensitive mutant strains were identified on YPD media containing 2 and 4 μg/ml of CTBT. Using pin replicator, cells of each mutant strain grown in YPD medium containing G418 sulphate were replicated as quadruplets to CTBT containing medium and to YPD control plate. After 6 days of growth the mutant strains sensitive to 2 or 4 μg/ml of CTBT were identified, collected and their sensitivity to CTBT confirmed in independent assays. This screen of the 4700 haploid gene deletion mutants was carried out once and resulted in the isolation of 169 CTBT hypersensitive mutant strains (Additional file 1).
Functions of selected genes deleted in CTBT sensitive mutant strains
Gene (ORF) name
ADH1, AFG3, ATP1, ATP11, ATP12, ATP18, CIT1, CYT1, DIA4, IMP1, ISA1, ISA2, MAS37, MDM32, MDM38, MGM1, MIP1, MRPL49, MRPS35, MRS1, MSY1, MTG1, OCT1, PCP1, PDC1, PHB1, RML2, RSM19, SOD1, SOD2, TUF1, YDR115W, YGL085W
Chromatin remodeling and transcription
ARP5, CDC73, CTK1, HOS2, HPR1, IES6, MED2, MGA2, OPI1, PGD1, RRN10, RME1, ROX3, RSC1, RTF1, RTT109, SGF73, SKN7, SNF2, SPT4, SPT20, SRB5, STP1, SWI3, SWI4, TAF14, THO2, UME6, YAF9, YAP1, YAP7
MET18, MMS1, MMS4, MRE11, RAD6, RAD18, RAD50, RAD51, RAD54, RAD57, XRS2
AKR1, CHO1, CHO2, DAP1, ERG2, ERG3, ERG6, ERG24, MGA2, OPI1
Stress response and signal transduction
ASC1, BCK1, CCS1, CTR1, CYS3, NBP2, REG1, SKN7, SNF1, SOD1, SOD2, YAP1, YAP7
AVT4, CWH36, KCS1, TFP1, TFP3, VMA4, VMA21, VMA22
Protein sorting and degradation
DIA2, MAP1, PRE9, RAD6, VPS15, VPS20, VPS34
Amino acid metabolism
CYS3, ILV1, PRO2, TRP2, TRP3, TRP5
AVT4, CTR1, MUP1, TAT1
Pentose phosphate pathway
ARD1, BEM1, BIM1, BUB3, BUD25, BUD27, BUR2, CDC50, CIK1, CSM1, CTF18, ENV6, FYV10, GEP4, GET2, HTZ1, KRE28, MTC5, NAT1, NAT3, NCE101, NPT1, NRP1, NUP133, ORM2, PHO85, RAI1, RCY1, REF2, RGI1, RNR4, RPL1B, RPL2A, RPL42B, RTC1, SBH1, YIM2
YDR049W, YDR114C, YHR045W, YNR065C, YOR305W
GO-terms significantly enriched in the 169 genes required for CTBT tolerance (SGD GO-termFinder).
response to stimulus
response to stress
organelle organization (mitochondrion)
The largest group of strains hypersensitive to CTBT contained deletions in genes for mitochondrial biogenesis and functions, including DNA replication (MIP1), mRNA processing (MRS1), protein synthesis and processing (AFG3, DIA4, MRPL49, MRPS35, MSY1, MTG1, OCT1, PCP1, RML2, RSM19, TUF1), respiration (CYT1), ATP synthesis (ATP1, ATP11, ATP12, ATP18), Fe/S protein biosynthesis (ISA1, ISA2), superoxide dismutation (SOD1, SOD2) and others.
In the second largest group were mutants in genes involved in gene expression thus hinting at an acute transcriptional response to CTBT. Identified genes are involved in chromatin remodeling (ARP5, HOS2, HTZ1, RSC1, SGF73, SNF2, SWI3, SWI4, YAF9), transcription (CTK1, MED2, ROX3, RRN10, RTF1, SPT4, SPT20, SRB5, TAF14, THO2) or encode transcription factors involved in oxidative stress response (YAP1, YAP7, SKN7) and lipid biosynthesis (OPI1).
We identified at least 11 CTBT sensitive strains containing deletion in DNA repair genes, including those involved in homologous recombination and repair (MMS1, MMS4, RAD50, RAD51, RAD52, RAD57), post replication repair (RAD6, RAD18), double strand break repair (MRE11, XRS2), excision repair (MET18) and others. Importantly, deletion of genes encoding functions in lipid metabolism also impaired CTBT tolerance. These are involved in ergosterol (DAP1, ERG2, ERG3, ERG6, ERG24), fatty acid (MGA2) and phospholipid biosynthesis (CHO1, CHO2, OPI1). Along with superoxide dismutase encoding genes, SOD1 and SOD2, which have a primary role in superoxide radical detoxification, other genes involved in oxidative stress response were also identified. The CCS1 gene encodes the specific copper chaperone delivering the copper to Sod1. The CTR1 gene is coding for a high affinity copper transporter of the plasma membrane. The transcription factors YAP1, YAP7 and SKN7 are involved in transcriptional regulation of oxidative stress response genes including SOD1, SOD2 and others. Additional functions required for CTBT tolerance involve vacuolar metabolism, protein sorting, amino acid metabolisms and others (Table 1). Importantly, many genes with CTBT defense functions overlap with those involved in menadion, hydrogen peroxide and arsenic stress tolerance [20–23].
The results of phenotypic profiling and the oxygen dependence of CTBT action, led us to the suggestion that CTBT induces reactive oxygen species in yeast cells.
Transcriptional profile analysis of CTBT treated yeast cells
GO-terms significantly enriched in the 500 > 2 fold induced or repressed genes (SGD GO-termFinder).
RNA polymerase complex
response to oxidative stress
monosaccharide catabolic process
response to chemical stimulus
cell redox homeostasis
alcohol metabolic process
Transcription factor binding sites enriched in CTBT regulated genes.
Mitochondrial superoxide production and petite mutant formation in CTBT treated yeast cells
Respiration deficient mutant formation in yeast cultures grown for 24 h in YPD medium containing indicated concentrations of CTBT
Petite mutants (%)
Theoretical treatment of CTBT activity
In order to get more insight into the mechanism of CTBT induced superoxide generation we used the standard computational protocol to perform quantum chemical calculations of 6 tetrazolo- and triazolobenzotriazines described previously . To compare the important structural parameters and their influence on the biological activity, the unsubstituted [1, 2, 4] triazolo [3,4, c]benzo[1,2,4]triazine (compound 3) was added to the original series. Four parameters that could be related to biological activity of compounds were chosen. LogP models the transport of molecules in biological systems, μ is the dipole moment of isolated molecule in Debye units, HOMO (Highest Occupied Orbital) and LUMO (Lowest Unoccupied Orbital) stand for energy of frontier orbitals in eV units.
In the chemical structure of studied molecules there are two structural characteristics that decrease significantly the LUMO-energy i.e. chlorine atom and tetrazolo ring that both are incorporated into CTBT. Consequently, CTBT appears as the main candidate for redox cycling and superoxide generation among the studied molecules. These calculations and the genetic data show that CTBT has a capacity to generate superoxide radicals with reducing equivalents possible derived from the respiratory chain.
In this study we show that CTBT, a compound enhancing the antifungal activity of several drugs , generates superoxide and other reactive oxygen species (ROS) and induces massive oxidative stress in yeast cells which enhances the antifungal activity of several unrelated drugs.
Five lines of evidence suggest that CTBT produces oxidative stress via generation of superoxide. First, CTBT toxicity required molecular oxygen. Second, it has predicted molecular properties of a molecule capable of redox cycling. Third, we detected oxidative stress using the two in vivo reporters MitoSOX Red and Yap1-GFP. Fourth, genetic evidence was provided by the isolation of characteristic mutants with defects in oxidative stress scavenging functions. Fifth, transcription profiling showed activation of regulons associated with oxidative stress response. CTBT activity was strictly dependent on the presence of molecular oxygen because no inhibition of growth by this drug was observed under strictly anaerobic conditions.
Antifungal activity of CTBT was higher on media containing glycerol plus ethanol instead of glucose indicating that developed functional mitochondria might be involved in drug action. This implies that apart from superoxide anion radical (O2.-) and ROS generation CTBT does not have other direct cytotoxic effects. ROS affect many cellular functions by damaging nucleic acids, oxidizing proteins and causing lipid peroxidation [35, 36]. Dismutation of superoxide into hydrogen peroxide and molecular oxygen is catalyzed by two superoxide dismutases: the Cu, Zn-depending Sod1p localized in the cytosol and the mitochondrial intermembrane space and the Mn-depending Sod2p which is localized in the mitochondrial matrix . Among the gene deletion strains selected for increased CTBT sensitivity (Additional file 1), the sod1Δ and sod2Δ mutant strains were found to be the most sensitive. Additionally, the mutants deleted for SOD1 and CCS1, a copper chaperone essential for Sod1p maturation, had a similar phenotype. CTBT could act as an inhibitor of Sods. Since the sod1Δsod2Δ double mutant cells were also sensitive to CTBT the Sod1p and Sod2p superoxide dismutases cannot be the primary targets of CTBT action. Therefore, CTBT is a producer of superoxide in presence of oxygen.
Most identified genes required for increased CTBT susceptibility were found to be involved in mitochondrial biogenesis and function, DNA repair, gene expression, lipid metabolism and stress response (Table 1). Many of them are known to be involved in defense processes protecting yeast cells against oxidative stress [35, 37] and have been previously identified in genome-wide analyzes of yeast deletion mutant strains sensitive to oxidative stress induced by a superoxide generator menadione, hydrogen peroxide, organic peroxides [20–22], arsenite and cadmium .
Remarkable is a high frequency of CTBT hypersensitive deletion mutants with affected mitochondrial functions. A requirement of energy for the repair of oxidatively damaged molecules has previously been proposed to explain why petite mutants are more sensitive to oxidative stress than wild type strains . On the other hand one cannot rule out a higher permeability of mitochondrial membranes for CTBT, superoxide or other ROS generated in dysfunctional mitochondria. The increased damage to mutant mitochondria caused by ROS may also reduce the mitochondrial membrane potential under critical level resulting in the arrest of mitochondrial biogenesis required for growth of eukaryotic cells .
Other significant pathways involved in CTBT susceptibility were also identified by the presence of several genes involved in the same pathway or encoding the subunits of the particular cellular complexes. This concerns genes involved in the RAD52 and RAD6 epistasis groups of DNA repair, Paf1 complex of RNA polymerase II (CDC73, HPR1 and RTF1), protein sorting to vacuole (VPS15, VPS34), vacuolar ATPase (TFP1, TFP3, VMA4, VMA21, VMA22), N-terminal acetyltransferases (ARD1, NAT1, NAT3) acetylating many proteins involved in cell cycle, heat shock resistance, mating, sporulation and telomere silencing as well as genes involved in ergosterol metabolism (DAP1, ERG2, ERG3, ERG6 and ERG24) and tryptophan biosynthesis (TRP2, TRP3 and TRP5). Along with tryptophan, interruption of the synthesis of cystein, isoleucin and proline also enhanced the CTBT toxicity. The sensitivity displayed by corresponding mutant strains is apparently not the result of the absence of the amino acids because CTBT was toxic on YPD plates that contain all necessary amino acids. It is possible that the accumulation of intermediates enhances the effect of CTBT.
The overlay of expression data with phenotypic data pointed mainly to superoxide dismutase activity (Sod1, Sod2, Ccs1) and second to the activation of transcription factors Yap1, Cin5/Yap4, and Yap7. Yap1p is a transcription activator involved in the control of multidrug resistance and oxidative stress response . Reactive oxygen species (ROS) generated both from endogenous and exogenous sources induce accumulation of Yap1p in the nucleus resulting in enhanced transcription of many genes involved in removing or detoxifying ROS. Cin5/Yap4 is activated by oxidative stress  like Yap1. The contribution of Yap7 to transcription is less understood. Yap7 is involved in transcriptional activation of the SOD1, SOD2 and CCS1 genes (http://yeastract.com;). However, we found no transcriptional induction of DNA damage specific genes and possibly because this stress type does not surface within the observed time frame. These findings suggest a highly focused primary effect of CTBT on oxidative stress and delayed effects on other pathways.
CTBT enhances activity of several drugs . This synergy becomes perhaps clearer when considering the production of superoxide and other ROS. Our phenotypic screen showed an enhanced sensitivity of mutants in the RAD52 and RAD6 epistasis groups of DNA repair. 5-Fluorocytosine is a drug which enters nucleotide metabolism and damages the cells by interfering with dNTP and mRNA synthesis. CTBT could act at two levels. Oxidative damage might cause DNA damage and at the same time hamper deoxynucleotide synthesis requiring glutathione or thioredoxin for production via ribonucleotide reductase. Azoles and terbinafin both target ergosterol synthesis. Interestingly, CTBT reduces transcription of most genes for the enzymes of the pathway. Finally, CTBT might exacerbate cycloheximide inhibition on translation by reduction of synthesis of ribosomal protein genes. Oxidative stress causes inactivation of the target of rapamycin complex 1 (TORC1) and thus inactivation of the Sfp1, one major activator of transcription of ribosomal protein genes. An interaction with the popular echinocandines remains to be shown. Up to now the combination of antifungals has been tried in vitro in many different combinations. The application of combinations may reduce costs, and importantly shift the effect of the drug towards fungicidal activity (for review see ). Apart from combinations of classical antifungals (amphotericin B, azoles, echinocandines), unusual combinations lead to unexpected results as for example in the case of azoles plus calcineurin inhibitors  or with membrane active compounds .
CTBT, apart from its weak antifungal activity, is able to strongly inhibit the proliferation of multidrug resistant yeast cells in combination with subinhibitory concentrations of other antifungals. Its mode of action depending on the molecular oxygen has been resolved using the combination of two genome-wide approaches including the screening of yeast deletion library for CTBT hypersensitivity mutants and transcriptome analysis of yeast cells exposed to this drug. We found that CTBT induces an increased production of superoxide and oxidative stress associated with damage to mitochondria and genomic DNA. Yeast cells deleted in nonessential genes encoding proteins involved mainly in mitochondrial function, DNA repair, transcription and oxidative stress response are hypersensitive to CTBT. CTBT rapidly induces transcription of oxidant and stress response defense genes activated mainly by Yap1 and Yap4/Cin5 transcription factors.
The exact molecular mechanism of CTBT action, associated with superoxide generation in mitochondria, is not known so far. It does not require a complete and functional respiratory chain as demonstrated by CTBT sensitivity of rho- mutant cells. Theoretical treatment of CTBT activity revealed that this compound might be amenable to one electron reduction. Electrons donated from mitochondrial NADH dehydrogenases or cytochrome bc1 complex can lead to CTBT anion radical formation that can be re-oxidized by molecular oxygen generating superoxide probable on the both sides of the inner mitochondrial membrane (Figure 7). Our combined genome wide approaches show the power of yeast genetics and transcript profiling to define mode of functioning of bioactive substances.
Strains and culture conditions
The following yeast strains were used:S. cerevisiae strains FY1679-28C (MATa ura3-52 trp1-63 leu2-1 his3-20) , BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), BY4742 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), the complete set of deletion mutants derived from the haploid strain BY4741 (EUROSCARF, http://web.uni-frankfurt.de/fb15/mikro/euroscarf), EG103 (MATα leu2-3,112 his3Δ1 trp1-289 ura3-52), EG118 (EC103 with sod1ΔA::URA3), EG110 (EC103 with sod2::TRP1), EG133 (EC103 with sod1ΔA::URA3 sod2::TRP1) . A plasmid expressing an N-terminal GFP-Yap1 fusion was obtained from M. Toledano . Cells were grown in YPD medium containing 2% (w/v) glucose, 1% (w/v) yeast extract, 2% (w/v) peptone, in YPGal medium (as YPD but 2% (w/v) galactose instead of 2% glucose), in YNB medium containing 2% (w/v) glucose 0.67% yeast nitrogen base without amino acids (Difco), in YPGE medium (as YPD but 2% glycerol plus 2% ethanol instead of 2% glucose). The media were solidified with 2% (w/v) bacto agar. Where appropriate, amino acids, uracil, ergosterol (20 μg/ml), Tween 80 (0.06%, w/v) or G418 sulphate (200 μg/ml) was added. For induction of rho-/rho0 mutants, cells were grown in YPD containing of ethidium bromide (25 μg/ml) or CTBT for 24 h, diluted and plated onto solid YPD. Frequency of respiration deficient mutants in yeast culture was determined after staining colonies with TTC (triphenyltetrazolium chloride) or replica plating onto YPGE plates.
Drug susceptibility testing
Susceptibility of yeast cells to CTBT was determined using the spot test assay. Aliquots of yeast cultures were spotted onto YPD plates containing the indicated concentrations of CTBT. Plates were incubated at 30°C for 6 days. In liquid media, susceptibilities to CTBT were assayed by the broth microdilution method in 96 well plate containing 200 μl YPD supplemented with different concentrations of CTBT. The growth at 30°C was scored after 24 and 48 h. Susceptibility to CTBT was also assessed using zone inhibition assays. Approximately 107 cells were plated onto YPD media, the filter discs (diameter of 6 mm) soaked with indicated amounts of CTBT were placed on the plates which were incubated at 30°C for 3-6 days before determination of the diameter of the zone of growth inhibition.
Screening for altered CTBT susceptibility
The collection of viable gene deletion mutants in the BY4741 background was screened for both CTBT hypersensitive and CTBT resistant strains. EUROSCARF mutant strains were transferred from 96 well master plates to solid YPD media supplemented with G418 sulphate. After 3 days, cells from grown colonies were inoculated into the corresponding wells of a 96 well microtiter plates containing 200 μl YPD supplemented with G418 sulphate. Cells were cultured 24 h at 30°C, diluted 20-times in YPD medium containing G418 sulphate and replica pinned onto YPD control plates and plates containing different concentration of CTBT (2 and 4 μg/ml) using a 96 floating pin replicator. The mutant strains were arranged in quadruplet to create a dilution in a given square giving a total of 96 strains plated per agar plate. The plates were incubated at 30°C and scored after 3 and 6 days. The altered sensitivity of strains to CTBT was assessed visually from the growth on the test medium relative to the growth on YPD control plate. To pin-point cellular functions that confer altered CTBT susceptibility, we searched for functional categories in the sensitive gene set according to FunCat at MIPS http://mips.gsf.de. Gene ontology (GO) analysis was done using GO Term Finder in SGD http://yeastgenome.org.
Wild type BY4741 cells were grown for 4 generations in YPD at 30°C to OD600 of 1 before CTBT solution (2 mg/ml) was added to a final concentration of 2 μg/ml. After 5, 10, 20 and 40 minutes cells were harvested, washed in ice-cold water and immediately frozen. RNA was isolated by the hot phenol method. 20 μg of total RNA was used for direct labeling cDNA synthesis with either Cy3-dCTP or Cy5-dCTP. Labeled cDNAs were purified with GFX columns (GE Healthcare). Hybridization to cDNA microarrays (Ontario Cancer Institute, Toronto, Canada) was done in triplicates with color inversion in 60 μl DigEasyHyb solution (Roche, Mannheim, Germany) overnight at 37°C. After hybridization, microarrays were washed three times in 1 × SSC, and 0.1% SDS at 50°C for 10 min, followed by 1 min in 1 × SSC und 0.1 × SSC at room temperature and 5 min 500 rpm spin to dryness. Microarrays were analyzed on an Axon 4000B scanner (Invitrogen, Molecular Devices) with Gene Pix Pro 4.1 (Axon; Molecular Probes).
For individual microarrays the intensity of the two fluorescent channels were normalized to the mean of ratio of medians of all unflagged features using the Genepix Pro 4.1 normalization option. Values of not found features were excluded from further analysis. Genes labeled as dubious ORFs in SGD were also removed from analysis. Mean ratios were calculated for features with at least 4 values. The filtered normalized values used for further analysis are available as supplementary file. Cluster analysis [51, 52]http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/software.htm was performed using the cluster3 and visualized with TreeView http://jtreeview.sourceforge.net. Significant associations to either GO-terms or transcription factors were obtained by GO-Term Finder at SGD http://www.yeastgenome.org and T-Profiler http://www.t-profiler.org. TreeView files corresponding to the figures are supplied as Additional files 3, 4, 5, 6, 7, 8. Values of genes associated with the most significant terms were visualized by Cluster analysis using complete linkage and correlation as similarity metric. GO assignments were graphically included in the cluster analysis by setting their column weight value to zero. Microarray data have been deposited at ArrayExpress http://www.ebi.ac.uk/microarray with the accession E-MEXP-2307.
Fluorescence microscopy and spectrometry
Intracellular ROS production was examined using MitoSOX Red (Molecular Probes). MitoSOX Red is a lipid soluble cation that accumulates in the mitochondrial matrix where it can be oxidized to a fluorescent product by superoxide . Yeast strains from initial concentration of 2 × 106cells/ml were grown in YPGal medium containing indicated concentration of CTBT. After 12 h of growth aliquots of 109 cells were washed twice with phosphate-buffered saline (PBS) and incubated in the dark for 20 min in 5 μM MitoSOX Red. Cells were washed three times with PBS, resuspended in PBS and the percentage of cells positively stained with MitoSOX Red was determined by fluorescence microscopy using a Zeiss Axioplan 2 fluorescence microscope (Thornwood, NY). Images were recorded on fluorescence microscope with a Spot Pursuit camera (Visitron Systems, Puchheim, Germany). Fluorescence of cells was also determined using fluorescence spectrometer (Jasco FP-6300, Tokyo) with excitation and emission wavelengths of 510 and 580 nm, respectively. Nuclei were stained by addition of 1 μl/ml Hoechst 33342 (Molecular Probes). GFP was visualized in live cells approximately 5 minutes after treatment with CTBT without fixation using excitation and emission wavelengths of 355 and 465 nm, respectively.
Quantum chemical calculations
The usual computational protocol for quantum chemical calculations was used. The optimal geometries of the molecules were obtained by complete geometry optimization employing the AM1 method. This geometry was used as input for the single SCF calculations by the ab Initio method (minimal STO-3G basis set) to obtain the energies and wave functions .
green fluorescent protein
highest occupied orbital
lowest unoccupied orbital
nicotinamide adenine dinucleotide (phosphate)
open reading frame
phosphate buffered saline
reactive oxygen species
sodium dodecyl sulfate
Saccharomyces genome database
saline-sodium citrate buffer.
We thank P. Griac for help with mutant screening, E. Gralla for strains, M. Toledano for material. The contribution of the Vienna FH-Biotech students to microarray analysis is acknowledged. We thank Gy. Hajos for providing the benzotriazine derivatives. MB was supported by grants from Comenius University (Grant UK 286/09) and FEMS (Research Fellowship FRF 2009-1). VK and IH were supported by grant from the Slovak Research and Developmental Agency (VVCE-0064-07-01). CS was supported by the Austrian Science Fund (FWF) grant B12-P19966, the UNICELLSYS FP7 grant (to Gustav Ammerer) and the Herzfelder Foundation. JG was supported by FWF grants P20444, P18955 and F34. JS was supported by grants from the Slovak Research and Developmental Agency (LPP-0022-06, LPP-0011-07, VVCE-0064-07-04) and Slovak Grant Agency of Science (VEGA 1/0001/09).
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