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Yeast Yeast 2011; 28: 81–91. Published online 22 October 2010 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.1825

Research Article

Chlorophenol stress affects aromatic amino acid biosynthesis — a genome-wide study Vikas Yadav, Kirti Shitiz, Rishi Pandey and Jyoti Yadav* Institute of Genomics and Integrative Biology (IGIB), CSIR, Delhi, India

*Correspondence to: Jyoti Yadav, Institute of Genomics and Integrative Biology (IGIB), Council of Scientific and Industrial Research (CSIR), Mall Road, Delhi 110007, India. E-mail: [email protected]

Received: 17 June 2010 Accepted: 7 September 2010

Abstract Chlorophenols are a class of chemicals commonly used in preservatives, disinfectants, algaecides, herbicides and pesticides. However, there is a growing evidence that these compounds are a threat to human health. This is alarming as many chlorophenols are common pollutants found in the global environment at potentially biohazardous levels. Despite chlorophenols being abundant, widely used and poisonous, we know relatively little about their mechanism of toxicity in eukaryotes. Thus, we performed genome-wide growth screens using Saccharomyces cerevisiae to understand the molecular basis of chlorophenol toxicity. Of ∼4850 single gene knockout strains tested, 393 mutants showed growth sensitivity to treatment with 4-chlorophenol (4-CP), 2,4dichlorophenol (2,4-DCP) or pentachlorophenol (PCP). Only eight mutants showed growth hypersensitivity to all the three treatments and harboured deletions in genes important for aromatic amino acid biosynthesis (ARO1, ARO7 ) or mitochondrial protein synthesis and respiration (ATP5, ISA1, RML2, GET2, SLS1, MRPL38 ). Copyright  2010 John Wiley & Sons, Ltd. Keywords: chlorophenol stress; Saccharomyces cerevisiae; aromatic amino acid biosynthesis; yeast deletion mutants

Introduction Chlorophenols are an important class of chemicals widely used in a variety of industrial processes. Among monochlorophenols, p-chlorophenol is commonly used as a disinfectant in dental practice (Hagiwara et al., 2006; Miyachi et al., 2005), whereas 4-chlorophenol (4-CP) is mainly used as a wood preservative in the synthesis of pesticides, fungicides and herbicides. It is also used for the bleaching of pulp with chlorine and in the disinfection of drinking water (Kim et al., 2002; Saez et al., 1993; Yuan et al., 2005). 4Chlorophenol is also a starting material for making germicides such as 2-benzyl-4-chlorophenol; it can also be converted to an analgesic, acetophenetidin (www.britannica.com). 2,4-Dichlorophenol (2,4-DCP) is also used as a wood preservative and in the synthesis of pesticides. It is an intermediate for the production of the herbicide Copyright  2010 John Wiley & Sons, Ltd.

2,4-dichlorophenoxy acetic acid (2,4-D) and other related herbicides, antibacterials such as triclosan, antihelminthic drugs etc. Exposure to 2,4-DCP could be fatal, as it is readily absorbed through the skin (Kintz et al., 1992; Proudfoot et al., 2003; Seiler et al., 1991). Pentachlorophenol (PCP), which was once the most extensively used pesticide, is used as a wood preservative against fungal rot and wood-boring insects. The widespread use of mono-, di- and polychlorophenols has led to their significant presence in the environment as a pollutant (Agency for Toxic substances, Disease Registry, 2001; IARC, 1991; Kintz et al., 1992). PCP is now banned in many countries and is listed as one of the major pollutants in the USA and Europe, mainly because of slow and incomplete biodegradation as well as improper disposal (Chen et al., 2004; Gupta et al., 2002; Wang et al., 2000). Chlorinated phenols are acidic in nature, and acidity increases as the degree of chlorination increases.

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The toxicity of chlorinated phenols directly relates to the number of chlorine atoms present in the molecule and it increases with increasing chlorination (Exon et al., 1984). Studies have shown that chlorinated phenols interfere with oxidative phosphorylation (Farquharson et al., 1958; Proudfoot et al., 2003). In vivo and in vitro experiments have suggested that PCP is a promoter of carcinogenesis in rodents (Chhabra et al., 1999), an endocrine disruptor and a probable carcinogen in humans (Proudfoot et al., 2003; Seiler et al., 1991). PCP has been shown to affect the endocrine system of vertebrate life forms (Beard et al., 1999) and is also reported to inhibit RNA and ribosome synthesis (Ehrlich et al., 1987). It causes adverse effects to the liver (Umemura et al., 1999; Kimbrough et al., 1978; Wang et al., 2001), kidneys, lungs, immune system (Blakley et al., 1998; Colosia et al., 1993) and gastrointestinal tract (Deichmann et al., 1942; George et al., 2006). Several in vivo (Yin et al., 2006) and in vitro (Wang et al., 2001; Wispriyono et al., 2002; Chen J et al., 2004; Chan X et al., 2004; Fernandez et al., 2005; Yang et al., 2005) studies regarding chlorophenol exposure have established that PCP-mediated toxicity is still little understood with regard to the molecular basis of the toxicity. Here, we try to understand the molecular mechanisms underlying the action of mono-, di- and polychloro-substituted phenols, using modern genome-wide screening techniques to address an old and very important biological problem. Using these modern techniques, we identified novel pathways involved in the mode of action of chlorophenol, along with confirmation of the previously reported role of mitochondrial genes. Saccharomyces cerevisiae, with the availability of an excellent genetic toolkit, represents an appropriate choice of eukaryotic model system (Winzeler et al., 1999) to study chlorophenol. A great degree of homology exists between yeast and humans (Foury et al., 1997). About 30% of the yeast genome has similarity with disease-causing human genes, which makes it even more suitable for genome-wide studies (Birrell et al., 2001). The availability of a systematically knocked-out collection of the whole genome makes it possible to identify genes whose deletion confers sensitivity to different chlorophenols. Copyright  2010 John Wiley & Sons, Ltd.

V. Yadav et al.

Materials and methods Yeast growth screen and analysis A library of 4848 Saccharomyces cerevisiae strains, with deletion of each non-essential gene in a haploid background (BY4741; MAT a), was purchased from Research Genetics (Resgen; Invitrogen, Carlsbad, CA, USA). 4-Chlorophenol was bought from Merck, Germany. 2,4-Dichlorophenol and pentachlorophenol were gifts from Dr Hemant Purohit of NEERI.

Growth assay The wild-type strain (BY4741) was pregrown in YPD liquid medium to mid-log phase, diluted to an optical density of 1.0 at 600 nm (OD600 nm ) and seeded in a 96-well microplate. Stock solutions of 4-CP, 2,4-DCP and PCP were added to the treatment wells in serially diluted concentrations, with at least three replicates/dose. The cells were then allowed to grow and the growth was monitored at 16 h using an Elisa Reader Spectramax (plus) from Molecular Probes, set to 30 ◦ C at OD600 nm . Raw absorbance data were averaged for all replicates, background corrected and plotted as a function of time.

Large-scale phenotypic screens Fifty-three 96-well microtitre plates representing the collection of mutant strains were thawed and 5 µl of each culture was used to inoculate 200 µl seed cultures grown for 14–19 h in YPD medium at 30 ◦ C. 5 µl of each seed culture was then used to inoculate 96-well microtitre plates, each containing 200 µl YPD medium with and without the addition of either 1.0 mM 4-CP, 0.3 mM 2,4-DCP or 0.156 mM PCP. Concentrations were chosen based on the results from serial dilution experiments with wild-type (WT). On each microtitre plate, two wells were reserved for a WT culture and for growth medium alone, representing positive and negative controls, respectively. Growth was monitored after incubation for 19 h at 30 ◦ C by measuring A600 nm on the Elisa Reader Spectramax, using Softmax Pro 4.8 software. Immediately prior to recording, the cultures were rapidly suspended using an electromagnetic plate shaker and all readings were made at 30 ◦ C. Yeast 2011; 28: 81–91. DOI: 10.1002/yea

Genome-wide chlorophenol screen

A600 nm values were background subtracted and normalized to average WT growth (mean calculated from 52 separate cultures spanning all microtitre plates per screen). Strains showing