1 United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 358 Saiwai, Fuchu, Tokyo 183; 2 Research Institute of Biological Resources, 3 Gene Function Research Center, 5 International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Higashi 11-1, Tsukuba, Ibaraki 3058566; 4 Daikin Environmental Laboratory, Ltd. 3 Banchi, Miyukigaoka, Tsukuba-shi, Ibaraki, 3050841 Japan
Received 8 October 2003; returned 6 December 2003; revised 15 March 2003; accepted 22 March 2004
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Abstract |
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Keywords: essential oils , ergosterol , stress response , toxicity , genomic expression
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Introduction |
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These plant-derived essential oils have been reported to show antimicrobial activity against a wide range of bacteria including antibiotic-resistant species.5,7 In addition to bactericidal or bacteriostatic activities, monoterpenes are also used to treat fungal infections, in particular genital and oral candidiasis, dermatophytoses, etc.2,8 All of these studies demonstrated that the activity of essential oils containing monoterpenes is both inhibitory and fungicidal. The antimicrobial activities and toxicity of monoterpenes (natural hydrocarbons) and other synthetic hydrocarbons have been well noted, but their modes of interaction with cells and the mechanism(s) of toxicity are largely unknown. Several studies concluded that, as lipophilic agents, they execute their action at the level of the membrane and membrane-embedded enzymes.9,10 Most recently, it has been reported that complete inhibition of Saccharomyces cerevisiae after exposure to palmarosa oil occurred due to a change in the fatty acid composition of the yeast cell membrane.3 Disruption of membrane integrity and the permeability barrier caused by tea tree oil was implicated as a mode of antimicrobial action against Candida albicans and different bacterial species.11,12 However, the response of a living cell to these compounds at the molecular level is not yet known.
To maintain the internal milieu at optimal conditions, a cell has to employ a specific genomic expression programme, in which a set of specific genes remains active whereas others remain switched off. When a cell faces a change in its surroundings caused by either a harmful chemical or drug, it reprogrammes its genomic expression to an adaptive response. Thus, measurement of changes in gene expression as an adaptive response upon exposure to a drug or chemical can help us to understand the mechanism of how drugs and drug candidates work in cells and organisms. DNA microarray analysis is powerful enough to provide a fast and systematic high-throughput analysis of gene expression at the level of the whole genome.13 S. cerevisiae is a good model organism for this type of analysis, because it adapts easily to changes in its environment and mimics many of the properties of higher organisms. Most notably, its genome sequence has already been completed and the functions of almost 70% of the genes are known, at least in part.14 Thus, the post-genomic era of S. cerevisiae has facilitated the identification of mechanisms of adaptive response of a whole organism to certain external or internal stimuli. Taking this advantage, yeast-based DNA microarrays have been used by our group and others to monitor global responses to environmental stresses and a variety of chemical agents with environmental health risk potential.1518 In addition, genomic profiling studies have been carried out to ascertain global responses to the toxicity of amphotericin B, 5-fluorocytosine and various azole compounds in both S. cerevisiae and C. albicans1922 at the molecular level. These studies have resulted in the development of a framework for predicting the mode of action of novel agents with antifungal activities.20,22 In this study, we report the global response of S. cerevisiae to -terpinene by monitoring the altered gene expression profiles using genome scale DNA microarrays.
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Materials and methods |
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S. cerevisiae strain S288C ( SUC2 mal gal2 CUP1) was used as an indicator strain for cDNA microarray analysis. It was grown in YPD medium (2% polypeptone, 1% yeast extract, and 2% glucose) at 25°C.
-Terpinene was purchased from Sigma Chemical Co. (USA). To optimize the culture conditions, exponentially growing cultures of yeast cells were exposed to various concentrations (0.0%, 0.01%, 0.02%, 0.03% and 0.04%) of
-terpinene for 24 h, and growth of the cells was measured every 2 h by counting colony forming units on YPD agar medium and also by measuring optical density at 660 nm (OD660). For transcriptional analysis, yeast cells diluted in YPD medium were incubated overnight at an OD of 1.0, and then 40 µL of terpinene was added to a 200 mL culture. After incubation for 2 h, cells were harvested by centrifugation and pellets were washed three times with DEPC-treated H2O before processing for RNA extraction.
Preparation of mRNA and cDNA probes
Total RNA was extracted by a hot-phenol method as described elsewhere.15 Poly(A)+ RNA was purified from total RNA with an Oligotex dT30 mRNA purification kit (Takara, Kyoto, Japan). Fluorescently labelled cDNA was synthesized by oligo(dT)primed polymerization using PowerScript reverse transcriptase (Clontech, CA, USA) in the presence of Cy3 (green) or Cy5 (red)-labelled deoxyuridine triphosphate (dUTP). The cDNA made from the poly(A)+ RNA of the control was fluorescently labelled with Cy3 and that of the terpinene-treated sample was labelled with Cy5. For each labelling, 24 µg of poly(A)+ RNA was used, and the same amount of each poly(A)+ RNA was used in one slide. For more details see Kitagawa et al.16
Hybridization and washing
Microarray hybridization was conducted using yeast DNA chips obtained from DNA Chip Research, Inc, Yokohama, Japan. The two labelled cDNA pools were mixed and hybridized with a yeast DNA chip for 2448 h at 65°C. When hybridization was complete, the labelled array was washed with 2 x SSC, 0.1% SDS, and with 0.2 x SSC, 0.1% SDS (twice for 20 min), and rinsed with 0.2 x SSC and 0.05 x SSC for 10 min each, and then dried.
Microarray analysis
Afterwards, labelled arrays were scanned with a confocal laser ScanArray 4000 (GSI Lumonics, Billerica, MA, USA) system. Array images were analysed with GenePix 4000 (Inter Medical). Responses to terpinene toxicity were determined by calculating the expression ratios of normalized Cy5 and Cy3 intensities. Normalization was carried out using the intensity of the median as the positive control, after reducing the intensities of the background and the non-specific signal. The background was the intensity around each spot, and the non-specific signal was the intensity due to solvent (spots with 10 mM TrisHCl 1mM EDTA, pH 8.0, buffer only). Gene Spring (Silicon Genetics, Redwood City, CA, USA) was used for further data analysis. For reliability of the data, changes in expression levels more than two-fold and <0.5-fold in at least two of the three independent experiments were considered to indicate induction and repression, respectively. The details of the microarray procedure have been described previously.16 The relative fold changes in the ratio of fluorescence intensity represent the average change in gene expression caused by the terpinene treatment. More than a two-fold increase was accepted as a basal level of induction and <0.5-fold was considered to be repression in the three independent experiments. We used at least two of the three in order to exclude genes that had high or low average values due to an irregularly high or low intensity in only one experiment.
Northern-blot analysis
Northern blotting was performed as described in Murata et al.17 Total RNA (20 µg) isolated from control or terpinene-treated cells was subjected to electrophoresis through 1.0% formaldehyde denaturing agarose gels (45 h at 100V). RNA was transferred to a nylon membrane (Roche Diagnostics) and fixed by UV cross-linking (120 mJ). Blots were probed with the double-strand DNA probes of the significantly induced genes, for example, INO1 and OPI3. Probes were made by PCR amplification using the chromosomal DNA as the template and the following primers:
INO1, 5'-TCTGCAACAACGCTTGAAGGGG-3' as the forward primer and 5'-AGCCATTCACCGGGTGAAATCC-3' as the reverse primer; OPI3, 5'-ATGAAGGAGTCAGTCCAAGAGATCA-3' as the forward primer and 5'-CATATTCTTTTTGGCCTTATCACGG-3' as the reverse primer; ACT1, 5'-TAACGGTTCTGGTATGTGTAAAGCC-3' as the forward primer and 5'-TGTAAGTAGTTTGGTCAATACCGGC-3' as the reverse primer. Each denatured probe was hybridized to the membrane-bound RNA, and detected with anti-digoxigenin antibody according to the manufacturer's instructions (Roche Diagnostics).
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Results |
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The purpose of this study was to assess the response of yeast cells to -terpinene at the molecular level. To this end, at first we optimized the concentration of terpinene that inhibited growth by
50% because strong or weak inhibition may cause undetectable responses. We found that 0.02% terpinene is enough to exert approximately half-maximal growth inhibition (IC50) (data not shown). Based on previous reports in which one doubling time (90 min) was sufficient to detect specific gene expression changes in response to various antifungal drugs,2022 yeast cells were treated with 0.02% terpinene for 2 h and RNA was prepared for microarrays from both terpinene-treated and untreated cells. DNA arrays of almost all of the yeast open reading frames were hybridized with Cy5- and Cy3-labelled probes, as described above. In Figure 1 the normalized signal intensities of the terpinene-treated samples are plotted against the normalized signal intensities of the controls. Data points shown in the top left and bottom right regions represent those ORFs that were either induced or repressed by terpinene treatment, respectively. Thus, a total of 793 genes were identified as responsive genes, and of them, 435 genes responded with increases in transcript levels upon terpinene treatment (the most significantly induced genes are shown in Table 1), and 358 genes were shown to respond with decreased mRNA levels (not shown).
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All of the responsive genes were annotated using the biological roles assigned by Munich International Centre For Protein Sequences (MIPS), and major representative classes of responsive genes were categorized according to their biological function as shown in Figure 2. We found that reprogramming of the genomic programme initiated the expression of proteins and enzymes related to lipid and fatty acid metabolism, transport facilitation, amino acid metabolism etc. in terpinene-treated cells. As was observed with commonly used antifungal drugs, we also clearly found that cell wall- and membrane-related genes were major targets of terpinene (Figure 2a). Notably, about 192 (44%) of the induced genes have not yet been functionally characterized, and their characterization will probably be helpful in understanding the mechanisms of antifungal drug resistance and sensitivity more clearly. Functional analysis of repressed genes showed that genes belonging to protein synthesis, carbohydrate metabolism and transcription categories were abundant among those genes that were repressed in terpinene-treated cells (Figure 2b).
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Microarray data revealed a global up-regulation of ERG genes along with other linked genes. This is in agreement with earlier studies showing that the ergosterol biosynthesis pathway is the target of azole derivatives in S. cerevisiae and its close relative C. albicans.19,21,22 Similar to these studies, a set of genes, e.g. ERG1, ERG3, ERG5, ERG6, ERG24, ERG28 (Figure 3), involved in the ergosterol biosynthesis pathway were transcriptionally activated in our experiment. In addition to the ergosterol biosynthesis genes, several other genes involved in this pathway also responded to terpene toxicity (Figure 3). For instance, overexpression of CYB5, which encodes cytochrome b5-reductase, was shown to cooperate with the ergosterol pathway downstream of ERG11. Thus, overexpression of CYB5 may reduce the sensitivity of S. cerevisiae to terpinene, and this is consistent with previous observations reported with azole compounds.21,23 Notably, and in agreement with previous studies,20,21 NCP1, which encodes NADP-cytochrome reductase, was also induced in this study. This enzyme acts as the electron donor for the products of ERG1, ERG11 and ERG5. Overexpression of SAM2 (S-adenosyl methionine synthetase), which catalyses the formation of S-adenosylmethionine (SAM) from methionine, also indirectly helps the ergosterol pathway by providing the substrate for ERG6 activity.21 In addition, FMS1, a multicopy suppressor of fenpropimorph resistance (Fen2 mutant) was up-regulated in response to terpinene action. In S. cerevisiae, fen2p was suggested to be a sensor of ergosterol levels in the membrane, thus allowing the cells to adjust to the growth conditions.24
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Apart from the up-regulation of the ergosterol biosynthesis pathway, several other genes related to inositol and lipid biosynthesis have been shown to change expression upon terpinene treatment (Table 1). The gene with the largest increase in expression (58.2-fold) in response to terpinene toxicity was INO1. Inositol is the precursor of phosphatidylinositol (PI), which is an essential membrane component of S. cerevisiae that acts in the cellular signal transduction pathway.25 The inositol that is required for PI synthesis is endogenously produced from glucose-6-phosphate, and the reaction is catalysed by Ino1p (inositol-1-phosphate-synthase).26 In addition, cells can take up inositol from the medium by expressing two transporters ITR1 and ITR2.27 Gene expression profiles show that Itr1 expression is also highly responsive to terpinene treatment. Consistent with this finding, expression of INO4, a regulator of ITR1 expression, was also significantly increased. Furthermore, CHO2/PEM1 and OPI3/PEM2, which catalyse the three subsequent methylation steps in the phospholipid biosynthesis pathway,17 were up-regulated in response to terpinene stress. In addition, the transcript level of GIT1, which encodes a transporter for glycerophosphoinositol (GroPIns), was also increased upon terpinene treatment. GIT1 is used to transport GroPIns from the extracellular medium during inositol starvation28 in terpinene-treated cells (Table 1).
Genes associated with cell wall organization
We have also found that a group of induced genes in terpinene-treated cells are implicated in cell wall biogenesis including CRH1, CHS1, GSC2, AGA2, SCW10, PIR2 (HSP150), KRE1, KRE26, ECM4, ECM13, ECM17, DAN1 etc. (Table 1). Crh1, whose expression is regulated differently during the life cycle,29 is a novel member of a group of cell wall-related proteins, and encodes a putative glycosidase that plays an important role in cell wall organization and maintenance by remodelling the glucan.30 It is known that GSC1 (FKS1) and GSC2 (FKS2) encode ß 13 glucan synthase that plays important roles in glucan synthesis.31,32 Mutations in the genes encoding these two proteins are lethal and, therefore, they are assumed to be essential for yeast survival.33 Similarly, genes involved with synthesis of ß 16 glucan, such as KRE1, were also up-regulated. Increased levels of transcripts of GSC2 and KRE1 in terpinene-treated cells could suggest that terpinene targets cell wall glucan synthesis and consequently may alter cell morphology and integrity. Our observation regarding the up-regulation of CHS1 (which encodes chitin synthase 1) in response to terpinene stress is very interesting, because it has a function in cell wall repair.34 In addition, we found that some of the cell wall mannoprotein-expressing genes significantly responded in terpinene-treated cells. Some of the mannoproteins (for example, Cwp1 and Cwp2) are expressed to adapt with environmental stress. One of the most highly expressed genes in terpinene-treated cells is DAN1 (delayed anaerobic)a gene whose expression is induced in response to stresses, such as anaerobiosis.35 Consistent with this result, a regulatory factor controlling the expression of the DAN/TIR genes, UPC2, was also up-regulated 19,36 in our study. According to an earlier report,35 the expression of Cwp1 (the major mannoprotein of the cell wall) was down-regulated due to anaerobic growth conditions, probably created by the terpinene treatment.
Our genomic data also revealed that the transcript level of YSR3, which encodes dihydrosphingosine 1-phosphate (DHS1-P) phosphatase, was significantly elevated. DHS1-P is a phosphorylated long chain (sphingoid) base (LCBP) and has been shown to be associated with the heat shock response.37,38 A recent study has shown that the intracellular accumulation of these phosphorylated molecules results in growth inhibition in S. cerevisiae. To cope with this unfavourable condition, the cell induces the expression of genes encoding members of the phosphatase family, such as Ysr2p and Ysr3p, to dephosphorylate LCBPs to LCBs.37
Induction of genes associated with detoxification
Most of the cyclic hydrocarbons are non-polar lipophilic compounds. To be excreted from the cell or for their metabolism, these compounds have to be oxidized to more polar compounds.39 These oxidation reactions are primarily mediated by the superfamily of cytochrome P450s and thus help in xenobiotic metabolism.40 As mentioned above, CYP60, the second cytochrome P450 gene in yeast, was highly induced in terpinene-treated cells. Based on an earlier report, it is reasonable to argue that the observed induction of CYP60 in response to terpinene could be a mechanism of xenobiotic resistance.39 Consistent with this proposal, yeast cells induced the expression of the FMO genes, which encode dimethyline monooxygenase and catalyse the mono-oxygenation of phosphorus-, sulphur- and nitrogen-containing xenobiotics including drugs, pesticides and industrial pollutants.41 Similarly, overexpression of RTA1 might also help the cell to attain resistance against terpene-mediated toxicity. RTA1 encodes a membrane-spanning protein that was reported to render resistance against the antifungal drug 7-aminocholesterol.42 Interestingly, some plants such as tobacco express a type of protein, namely osmotin, belonging to the PR-5 family, that inhibits fungal growth as a defence mechanism.43 However, further studies showed that the fungus becomes resistant to osmotin by inducing a gene family (PIR) encoding the membrane-embedded stress protein Hsp150. Therefore, increased expression of HSP150 against terpene cytotoxicity could function as a mode of xenobiotic resistance in S. cerevisiae. Based on this observation, it could be hypothesized that plants produce terpinene to defend themselves from pathogenic fungi. These observations suggest that terpene compounds could be considered to be xenobiotics, and in response, the cell induces the expression of genes associated with drug metabolism and detoxification pathways.
Validation of microarray data by northern blotting
There have been arguments suggesting that microarray data may not always be reliable. To validate the differential expression of genes identified in the microarray experiments, the expression of selected genes can be tested either by northern-blot analysis or real-time PCR. In this study, the transcription of two abundantly induced ORFs identified in the microarray analysis, INO1 and OPI3, along with ACT1 (as a loading control), were verified by northern blotting. The results (Figure 4) are in complete agreement with the microarray experiments.
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Discussion |
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This study showed that global up-regulation of genes associated with the ergosterol biosynthesis pathway occurs in response to terpinene toxicity. This suggests that ergosterol synthesis was strongly inhibited in terpinene-treated cells. Interestingly, azole compounds such as fluconazole, miconazole and clotrimazole etc., commonly used as antifungals, target and inhibit the ergosterol biosynthesis pathway in C. albicans and S. cerevisiae. In addition to azoles, other classes of antifungal drugs, such as polyenes, allylamines and morpholines also target ergosterol biosynthesis as a mode of fungal growth inhibition. Most notably, similar microarray analysis conducted with these compounds19,21,22 also showed a global up-regulation of erg genes, as observed with terpinene in our study. But, in contrast to these studies, expression of cytochrome P450 (CYP51) encoding ERG11, a major target of azole compounds, was not increased in terpinene-treated cells. Instead, another cytochrome P450-encoding gene, ERG5 (CYP61), which encodes c-22 desaturase,44 was responsive. It is believed that ERG11 works under aerobic conditions when ERG3 is not functional.45 The observed induction of ERG3 in our study suggests that ERG11 function was silenced, probably due to the anaerobic conditions created by terpinene treatment. But we do not have any direct evidence that culture conditions were anaerobic. However, abundant induction of DNA1 and down-regulation of Cwp1 in terpinene-treated cells could be circumstantial evidence of anaerobic conditions.35,36
However, which mechanism is responsible for the up-regulation of the ERG genes in response to terpinene toxicity remains unknown. One study46 has shown that ergosterol limitation in S. cerevisiae induces the expression of ERG1. A study reported by DeBacker et al.21 suggested that sterol limitation may cause the up-regulation of ERG1 in C. albicans in response to itraconazole treatment. Additionally, expression of the ERG3 and ERG9 genes was also found to be regulated by ergosterol availability.20 Similarly, overexpression of ERG1 and ERG3 in our study indicates that sterol depletion in terpinene-treated cells may be the reason for up-regulation of ergosterol biosynthesis. This could be further supported by overexpression of FMS1, which acts as a sensor of ergosterol levels in the cell membranes. The other hypothesis argues that accumulation of toxic sterol by-products, due to the inhibition of specific steps in the ergosterol biosynthesis pathway, induces ERG expression.
An interesting observation from our study is the overexpression of several genes associated with sulphur assimilation, methionine biosynthesis and AdoMet production. MET6, MET17/25, MET8, MET13, MET14, MET10, SAM2, SAM3 etc. were found to be overexpressed in the presence of terpinene (Table 1). One explanation of this observation could be the continuous supply of a methyl group in the methylation reaction catalysed by ERG6 in the ergosterol biosynthesis pathway, and also that three subsequent methylation reactions in the phospholipid biosynthesis pathway (not shown) may have led to the activation of AdoMet biosynthesis. A similar observation was also reported in DMSO-treated yeast cells by Murata et al.17 Consistent with their study, we can also predict that overexpression of INO1 and OPI3, two important genes of phospholipid biosynthesis, and simultaneous activation of the methionine biosynthesis pathway, may induce lipid proliferation in terpinene-treated cells. This proposal has yet to be proven.
Another important observation from our study is the significant up-regulation of a large number of genes associated with cell wall biogenesis. We could see that cell wall mannoproteins, ß-glucan as well as chitin synthesis pathways were activated in terpinene-treated cells. It is well known that the complex structures of these three compounds define the yeast cell wall architecture, which is essential for the maintenance of cell shape, cellular integrity and protection against harmful environments.47 Obviously, perturbation or damage of cell wall structures caused by environmental insults or any cell wall-targeting drug must be deleterious to fungal growth. Yeast has developed mechanisms to compensate for these attacks and one of these mechanisms is termed cell wall compensatory mechanism.48 Three main responses have been identified to explain how cell wall stresses induce a change in cell wall arrangement.49 First, hyper-accumulation of chitin occurs to bring a change in cell wall polysaccharides. Second, a change in glucan and cell wall mannoprotein synthesis takes place to increase the mechanical strength of the cell wall, and third, cell wall synthesis and repairing machineries are redistributed all over the cell. Taking these into account, overexpression of a group of genes associated with cell wall biogenesis in terpinene-treated cells clearly indicates that it can affect cell wall structures, which in turn activates cell wall compensatory mechanism to overcome the stress.
Consistent with their lipophilic nature, many earlier studies have implicated the toxicity on membrane structures as a mode of antimicrobial action of essential oils and their monoterpenoid components.9,10,50 Very recently, Prashar et al.3 showed that the antimicrobial affect of palmarosa oil on S. cerevisiae led to a change in fatty acid composition of the yeast cell membrane, with more saturated and fewer unsaturated fatty acids in the membrane. Similarly, using a biochemical approach Cox et al.12 found that tea tree oil targets cell membrane permeability and fluidity as a mode of antimicrobial action against Gram-negative bacteria and the yeast C. albicans. In our study, functional analysis of induced genes by yeast DNA microarray allowed us to monitor the antimicrobial activities of a monoterpene on S. cerevisiae at a molecular level. It is clear from our data, in addition to the ergosterol and lipid biosynthesis pathway, that cell wall structures were severely affected by terpinene toxicity. Taken together, we could say that the mechanisms of antifungal action depicted by microarray data are very consistent with other mechanisms, as described above.
In conclusion, a global view of changes in gene expression in response to the antifungal action of terpinene was obtained with DNA microarrays. Analysis of these data revealed that specific changes in gene expression were consistent with mechanisms of action of other commonly used antifungal drugs. In addition, some non-specific changes were also observed, along with changes in the expression of several genes of unknown function. Obviously, understanding the function of these unknown genes, in addition to known genes, will probably be useful to identify more targets for the design of antifungal drugs. In addition, our data will be useful for characterizing the mechanisms of antifungal activities of many widely used folk medicines. These data could also be helpful in obtaining a clear understanding of the mechanisms of toxicity of other monoterpenes and cyclic hydrocarbons.
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Acknowledgements |
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Footnotes |
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References |
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2
.
Hammer, K. A., Carson, C. F. & Riley, T. V. (2002). In vitro activity of Melaleuca alternifolia (tea tree) oil against dermatophytes and other filamentous fungi. Journal of Antimicrobial Chemotherapy 50, 1959.
3 . Prashar, A., Hili, P., Veness, R. G. et al. (2003). Antimicrobial action of palmarosa oil (Cymbopogon martinii) on Saccharomyces cerevisiae. Phytochemistry 63, 56975.[CrossRef][ISI][Medline]
4
.
Nelson, R. R. (1997). In-vitro activities of five plant essential oils against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Journal of Antimicrobial Chemotherapy 40, 3056.
5
.
Elsom, G. K. & Hide, D. (1999). Susceptibility of methicillin-resistant Staphylococcus aureus to tea tree oil and mupirocin. Journal of Antimicrobial Chemotherapy 43, 4278.
6 . Carson, C. F. & Riley, T. V. (1995). Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia. Journal of Applied Bacteriology 78, 2649.[ISI][Medline]
7 . Inouye, S., Yamaguchi, H. & Takizawa, T. (2001). Screening of the antibacterial effects of a variety of essential oils on respiratory tract pathogens, using a modified dilution assay method. Journal of Infectious Chemotherapy 7, 2514.[CrossRef][Medline]
8 . Nenoff, P., Haustein, U. F. & Brandt, W. (1996). Antifungal activity of the essential oil of Melaleuca alternifolia (tea tree oil) against pathogenic fungi in vitro. Skin Pharmacology 9, 38894.[ISI][Medline]
9 . Uribe, S., Ramirez, J. & Pena, A. (1985). Effects of beta-pinene on yeast membrane functions. Journal of Bacteriology 161, 11951200.[ISI][Medline]
10
.
Sikkema, J., de Bont, J. A. & Poolman, B. (1994). Interactions of cyclic hydrocarbons with biological membranes. Journal of Biological Chemistry 269, 80228.
11 . Bard, M., Albrecht, M. R., Gupta, N. et al. (1988). Geraniol interferes with membrane functions in strains of Candida and Saccharomyces. Lipids 23, 5348.[ISI][Medline]
12 . Cox, S. D., Mann, C. M., Markham, J. L. et al. (2000). The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). Journal of Applied Microbiology 88, 1705.[CrossRef][ISI][Medline]
13 . Brown, P. O. & Botstein, D. (1999). Exploring the new world of the genome with DNA microarrays. Nature Genetics 21, 337.[CrossRef][ISI][Medline]
14 . Goffeau, A. (2000). Four years of post-genomic life with 6,000 yeast genes. FEBS Letters 480, 3741.[CrossRef][ISI][Medline]
15 . Parveen, M., Momose, Y., Kitagawa, E. et al. (2003). Bioassay of pesticide lindane using yeast-DNA microarray technology. Chem-Bio Informatics Journal 3, 1229.[CrossRef]
16 . Kitagawa, E., Takahashi, J., Momose, Y. et al. (2002). Effects of the pesticide thiuram: genome-wide screening of indicator genes by yeast DNA microarray. Environmental Science & Technology 36, 390815.[CrossRef][ISI][Medline]
17
.
Murata, Y., Momose, Y., Hasegawa, M. et al. (2003). Dimethyl sulfoxide exposure facilitates phospholipid biosynthesis and cellular membrane proliferation in yeast cells. Journal of Biological Chemistry 278, 3318593.
18
.
Gasch, A. P., Spellman, P. T., Kao, C. M. et al. (2000). Genomic expression programs in the response of yeast cells to environmental changes. Molecular Biology of the Cell 11, 424157.
19
.
Agarwal, A. K., Rogers, P. D., Baerson, S. R. et al. (2003). Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cerevisiae. Journal of Biological Chemistry 278, 3499835015.
20
.
Bammert, G. F. & Fostel, J. M. (2000). Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol. Antimicrobial Agents and Chemotherapy 44, 125565.
21
.
De Backer, M. D., Ilyina, T., Ma, X. J. et al. (2001). Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray. Antimicrobial Agents and Chemotherapy 45, 166070.
22
.
Zhang, L., Zhang, Y., Zhou, Y. et al. (2002). Response of gene expression in Saccharomyces cerevisiae to amphotericin B and nystatin measured by microarrays. Journal of Antimicrobial Chemotherapy 49, 90515.
23 . Truan, G., Epinat, J. C., Rougeulle, C. et al. (1994). Cloning and characterization of a yeast cytochrome b5-encoding gene which suppresses ketoconazole hypersensitivity in a NADPH-P-450 reductase-deficient strain. Genetics 142, 1237.
24 . Marcireau, C., Joets, J., Pousset, D. et al. (1996). FEN2: a gene implicated in the catabolite repression-mediated regulation of ergosterol biosynthesis in yeast. Yeast 12, 5319.[CrossRef][ISI][Medline]
25 . Daum, G., Lees, N. D., Bard, M. et al. (1998). Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14, 1471510.[CrossRef][ISI][Medline]
26 . Majumder, A. L., Johnson, M. D. & Henry, S. A. (1997). 1L-myo-inositol-1-phosphate synthase. Biochimica et Biophysica Acta 1348, 24556.[ISI][Medline]
27
.
Nikawa, J., Tsukagoshi, Y. & Yamashita, S. (1991). Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae. Journal of Biological Chemistry 266, 1118491.
28 . Patton-Vogt, J. L. & Henry, S. A. (1998). GIT1, a gene encoding a novel transporter for glycerophosphoinositol in Saccharomyces cerevisiae. Genetics 49, 170715.
29
.
Rodriguez-Pena, J. M., Cid, V. J., Arroyo, J. et al. (2000). A novel family of cell wall-related proteins regulated differently during the yeast life cycle. Molecular and Cellular Biology 20, 324555.
30 . Jung, U. S. & Levin, D. E. (1999). Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway. Molecular Microbiology 34, 104957.[CrossRef][ISI][Medline]
31 . Garrett-Engele, P., Moilanen, B. & Cyert, M. S. (1995). Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H(+) ATPase. Molecular and Cellular Biology 15, 410314.[Abstract]
32 . El-Sherbeini, M. & Clemas, J. A. (1995). Cloning and characterization of GNS1: a Saccharomyces cerevisiae gene involved in synthesis of 1,3-beta-glucan in vitro. Journal of Bacteriology 177, 322734.[Abstract]
33 . Mazur, P., Morin, N., Baginsky, W. et al. (1995). Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase. Molecular and Cellular Biology 15, 567181.[Abstract]
34 . Orlean, P. (1997). Biogenesis of yeast wall and surface components. Molecular Biology of the Yeast Saccharomyces 3, 229362.
35
.
Cohen, B. D., Sertil, O., Abramova, N. E. et al. (2001). Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites. Nucleic Acids Research 29, 799808.
36
.
Abramova, N. E., Cohen, B. D., Sertil, O. et al. (2001). Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae. Genetics 157, 116977.
37
.
Mandala, S. M., Thornton, R., Tu, Z. et al. (1998). Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response. Proceeding of the National Academy of Sciences, USA 95, 1505.
38
.
Skrzypek, M. S. & Nagiec, M. M., Lester, R. L. et al. (1999). Analysis of phosphorylated sphingolipid long-chain bases reveals potential roles in heat stress and growth control in Saccharomyces cerevisiae. Journal of Bacteriology 181, 113440.
39 . Kelly, S. L., Lamb, D. C. & Kelly, D. E. (1997). Sterol 22-desaturase, cytochrome P45061, possesses activity in xenobiotic metabolism. FEBS Letters 412, 2335.[CrossRef][ISI][Medline]
40 . Nelson, D. R., Kamataki, T., Waxman, D. J. et al. (1993). The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA and Cell Biology 12, 151.[ISI][Medline]
41 . Lawton, M. P., Cashman, J. R., Cresteil, T. et al. (1994). A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities. Archives of Biochemistry and Biophysics 308, 2547.[CrossRef][ISI][Medline]
42 . Soustre, I., Letourneux, Y. & Karst, F. (1996). Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance. Current Genetics 31, 1215.[CrossRef]
43
.
Yun, D. J., Zhao, Y., Pardo, J. M. et al. (1997). Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein. Proceedings of the National Academy of Sciences, USA 94, 70827.
44 . Arthington-Skaggs, B. A., Crowell, D. N., Yang, H. et al. (1996). Positive and negative regulation of a sterol biosynthetic gene (ERG3) in the post-squalene portion of the yeast ergosterol pathway. FEBS Letters 392, 1615.[CrossRef][ISI][Medline]
45
.
Sanglard, D., Ischer, F., Parkinson, T. et al. (2003). Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrobial Agents and Chemotherapy 47, 240412.
46 . M'Baya, B., Fegueur, M., Servouse, M. et al. (1989). Regulation of squalene synthase and squalene epoxidase activities in Saccharomyces cerevisiae. Lipids 24, 10203.[ISI][Medline]
47 . Klis, F. M., Mol, P., Hellingwerf, K. et al. (2002). Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiology Reviews 26, 23956.[CrossRef][ISI][Medline]
48
.
Bickle, M., Delley, P. A., Schmidt, A., & Hall, M. N. (1998). Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO Journal 17, 223545.
49
.
Lagorce, A., Hauser, N. C., Labourdette, D. et al. (2003). Genome-wide analysis of the response to cell wall mutations in the yeast Saccharomyces cerevisiae. Journal of Biological Chemistry 278, 2034557.
50 . Knobloch, K., Pauli, A., Iberl, B. et al. (1988). Antibacterial activity and antifungal properties of essential oil components. Journal of Essential Oils Research 1, 11928.