**1. Introduction**

Extensive research over the past decade has found the widespread presence of organic wastewater contaminants (OWC) in surface waters around the globe including the United States, (Alvarez et al., 2009; Focazio et al., 2008; Kolpin et al., 2002; Owens et al., 2007; Zheng et al., 2008), Asia (Ma et al., 2007), Europe (Cargouet et al., 2007; Cespedes et al., 2005; Gros et al., 2009; Reemtsma et al., 2006) and South America (Bergamasco et al., submitted; Jardim et al., 2011; Kuster et al., 2009). These OWC include pesticides, plasticizers, pharmaceuticals, and natural and synthetic hormones as well as pollutants from chemical spills into the environment. These compounds may be introduced into surface waters by runoff from land application of biosolids, through leaking sewer lines and septic systems, or by incomplete removal from wastewater treatment systems. Further, a wide variety of these chemicals have been implicated in endocrine disruption in invertebrates and vertebrates (Cooper & Kavlock, 1997; Fang et al., 2000; Folmar et al., 2002; Fossi & Marsili, 2003; Guillette et al., 1999; Hayes et al. 2010; Kavlock et al., 1996; Kidd et al. 2007; Ropstad et al., 2006; Sonne et al., 2006; Tyler et al., 1998).

An endocrine disruptor is an exogenous substance that causes adverse health effects in an organism or its offspring by way of alteration in the function of the endocrine system. As such endocrine disruption is a mechanism leading to a variety of adverse health effects, most of which are considered as reproductive or developmental toxicities (OECD, 2002). The complex nature of reproductive and developmental effects suggests that *in vivo* tests are necessary to detect endocrine disruption. Several i*n vivo* mammalian assays (e.g. O'Connor et al., 2002) and *in vitro* assays (e.g. Fang et al., 2000; Zacharewski, 1997) exist for measuring estrogenic effects in various biological systems. However, these are not suitable for rapid, high-throughput screening of chemicals or necessarily screening of environmental samples. Yeast-based *in vitro* estrogen and androgen screens have been firmly established as a means for rapidly identifying chemicals with potential endocrine disrupting activity. This chapter will review the development and use of yeast-based bacterial bioluminescent bioreporters for the detection of endocrine disruption compounds.

Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters 5

throughput analysis due to additional handling steps (costly substrate addition) and

The *luc* genes have been reported to be more sensitive than *lux*-based systems, however in a recent comparison of *luc*- and *lux*-based hormone-sensing bioreporters, Svobodova and Cajthaml (2010) determined that some *lux*-based bioreporters (BLYES/BLYAS bioassays, discussed below) are of comparable sensitivity and in some cases much more sensitive than

Several reviews are available on the properties and use of *luc*, *luxAB, luxCDABE, gfp,* and *gfp-*derived reporter genes in environmental systems (Hakkila et al., 2002; Keane et al., 2002; Ripp et al., 2010). Each of these reporter technologies has advantages and disadvantages depending on the application. For high throughput analysis of samples, bioreporters with the *luxCDABE* genes expressed are particularly well-suited for screening large numbers of samples. For both *luxAB*- and *lucFF*-based bioreporters, costly substrates must be continually added to the cells for visualization of the reaction. This increases not only handling difficulty but also costs to perform the assay. For GFP-based bioreporters, no exogenous substrates are necessary but fluorescent molecules must be excited by a light source to fluoresce. Each of these types of bioreporters produces signals for different lengths of time and has different light emission maxima and optimum temperatures. For example, while the *Photorhabdus luminescens* luciferase (Lux) is stable up to 42oC, firefly luciferase (Luc) has a temperature optimum at 25oC and is thermally inactivated above 30oC (Keane et al., 2002). Bioreporter fusions incorporating the full *lux* cassette are advantageous in that they do not require exogenous substrates, cell lysis is not required, the signal is quantitative and reproducible (King et al., 1990). Further, continuous on-line monitoring is possible (e.g. DiGrazia et al., 1991; Heitzer et al., 1994;

Prior to 2003, the *lux* genetic system was previously limited only to expression in prokaryotic systems. However, Gupta et al. (2003) were successful in expressing the *P. luminescens lux* cassette in the yeast *S. cerevisiae*. Specifically, the *luxA, -B, -C, -D*, and -*E* genes from *P. luminescens* and the *frp* gene from *Vibrio harveyi* were re-engineered for expression in *Saccharomyces cerevisiae*. The *lux* operon was engineered using two pBEVY yeast expression vectors (Miller et al., 1998), which allowed bidirectional, constitutive expression of the individual *luxA, -B, -C, -D*, and -*E* genes. The *luxA* and *luxB* genes were independently expressed from divergent yeast constitutive promoters GPD and ADH1 on pBEVY-U (Figure 1). The *luxCD* and *luxE-frp* genes were independently expressed from a second plasmid (pBEVY-L), also using the GPD and ADH1 promoters. An internal ribosome entry site (IRES) was inserted between the *luxC* and *luxD* genes and the *luxE* and *frp* genes. The IRES allows translation of multiple genes from a single promoter in eukaryotes (Hellen

Constitutive expression of the *luxCDABEfrp* genes in *S. cerevisiae* W303a generated approximately 9,000,000 photons per second per unit optical density (Gupta et al., 2003). This is comparable to similar expression in prokaryotic systems. This was a significant milestone in expression of bacterial operons in lower eukaryotic systems and created possibilities for screening organic wastewater contaminants with mammalian health

additional cost.

*luc*-based bioreporters.

Heitzer et al., 1992; King et al., 1990).

& Sarnow, 2001).

significance.

**1.2 Bacterial** *lux* **expression in** *Saccharomyces cerevisiae*

#### **1.1 Bioreporters**

Reporter gene fusions have been widely used for the detection and quantification of chemical, biological, and physical agents (Daunert et al., 2000). The principle is to fuse a specific genetic promoter or response element with a reporter gene. Induction by a specific target chemical initiates transcription/translation of the bioreporter molecule, which generates a measurable signal. There are three widely-used classes of bioreporters: colorimetric (e.g. *lacZ, cat*), fluorescent (e.g. *gfp*), and bioluminescent (e.g. *luc, lux*). One example of a colorimetric-based bioreporter is the *lacZ* gene which encodes the βgalactosidase enzyme. β-Galactosidase mediates the breakdown of lactose to glucose + galactose. As a bioreporter, β-galactosidase is widely used in molecular biology in the bluewhite screening assay. The chromophore X-gal (bromo-chloro-indolyl-galactopyranoside) is cleaved into galactose and an indole moiety that turns the medium blue. For chemical detection, *lacZ* is fused to a chemical-responsive promoter and when the cells are exposed to chromophores, such as chlorophenol red-β-D-galactopyranoside (CPRG), the assay medium changes from yellow to red. This type of colorimetric bioreporter is inexpensive and can be used in a qualitative or quantitative type of assay. Color density can be measured on a standard spectrophotometer.

Fluorescent assays take advantage of the green fluorescent protein (GFP). GFP was originally isolated from the jellyfish *Aequorea victoria* (Johnson et al., 1962; Shimomura et al., 1962). GFP is widely used as a bioreporter in eukaryotic systems for its simplicity to clone and no requirement for an organic substrate other than excitation with either UV or blue light. Quantification of the signal is by a fluorescent spectrophotometer or plate reader. There are different versions of *gfp* including blue-, red-, and yellow-shifted variants each requiring different excitation wavelengths and each of which fluoresce at different wavelengths (Hein & Tsien, 1996; Kendall & Badminton, 1998). In some cases this may be advantageous, especially when multiple bioreporters will be used simultaneously. These genes have been used extensively since they were first employed as gene expression biomarkers (Chalfie et al., 1994).

Firefly luciferase is another well-used bioreporter in eukaryotic systems. The luciferase, encoded by the *luc* gene (*lucFF*), was originally isolated from *Photinus pyralis* (firefly) and generates luciferase by a two-step conversion of D-luciferin to oxyluciferin (de Wet et al., 1985). This reaction generates light at 560 nm. However, the gene does not encode for the Dluciferin substrate and therefore substrate addition in any assay is required, which adds processing time and expense to the assay. Luc-based assays may also be constrained by the requirement for a cell lysis step followed by addition of the D-luciferin, adding both time and expense to the assay.

Bacterial bioluminescence has been widely used as a bioreporter in prokaryotic systems. The *lux* operon (*lux*CDABE) was originally isolated from *Vibrio fischeri* (Engebrecht et al., 1983), *Vibrio harveyi* (Cohn et al., 1983), and *Photorhabdus luminescens* (Szittner & Meighen, 1990)*.* The *lux* operon encodes for the luciferase enzyme (*luxAB*) and the long-chain aldehyde substrate (*luxCDE*) for that reaction. An assay employing bacterial bioluminescence does not require an external organic substrate; the only requirement is for oxygen (O2). A long chain aldehyde and a reduced flavin mononucleotide (FMNH2) are converted by luciferase (LuxAB) to a long chain carboxylic acid and FMN, producing light at 490 nm wavelength (Meighen & Dunlap, 1993). The *luxAB* (without *luxCDE*) can also be used as a bioreporter and while these strains also produce light at 490 nm, they are less suited for high

Reporter gene fusions have been widely used for the detection and quantification of chemical, biological, and physical agents (Daunert et al., 2000). The principle is to fuse a specific genetic promoter or response element with a reporter gene. Induction by a specific target chemical initiates transcription/translation of the bioreporter molecule, which generates a measurable signal. There are three widely-used classes of bioreporters: colorimetric (e.g. *lacZ, cat*), fluorescent (e.g. *gfp*), and bioluminescent (e.g. *luc, lux*). One example of a colorimetric-based bioreporter is the *lacZ* gene which encodes the βgalactosidase enzyme. β-Galactosidase mediates the breakdown of lactose to glucose + galactose. As a bioreporter, β-galactosidase is widely used in molecular biology in the bluewhite screening assay. The chromophore X-gal (bromo-chloro-indolyl-galactopyranoside) is cleaved into galactose and an indole moiety that turns the medium blue. For chemical detection, *lacZ* is fused to a chemical-responsive promoter and when the cells are exposed to chromophores, such as chlorophenol red-β-D-galactopyranoside (CPRG), the assay medium changes from yellow to red. This type of colorimetric bioreporter is inexpensive and can be used in a qualitative or quantitative type of assay. Color density can be measured on a

Fluorescent assays take advantage of the green fluorescent protein (GFP). GFP was originally isolated from the jellyfish *Aequorea victoria* (Johnson et al., 1962; Shimomura et al., 1962). GFP is widely used as a bioreporter in eukaryotic systems for its simplicity to clone and no requirement for an organic substrate other than excitation with either UV or blue light. Quantification of the signal is by a fluorescent spectrophotometer or plate reader. There are different versions of *gfp* including blue-, red-, and yellow-shifted variants each requiring different excitation wavelengths and each of which fluoresce at different wavelengths (Hein & Tsien, 1996; Kendall & Badminton, 1998). In some cases this may be advantageous, especially when multiple bioreporters will be used simultaneously. These genes have been used extensively since they were first employed as gene expression

Firefly luciferase is another well-used bioreporter in eukaryotic systems. The luciferase, encoded by the *luc* gene (*lucFF*), was originally isolated from *Photinus pyralis* (firefly) and generates luciferase by a two-step conversion of D-luciferin to oxyluciferin (de Wet et al., 1985). This reaction generates light at 560 nm. However, the gene does not encode for the Dluciferin substrate and therefore substrate addition in any assay is required, which adds processing time and expense to the assay. Luc-based assays may also be constrained by the requirement for a cell lysis step followed by addition of the D-luciferin, adding both time

Bacterial bioluminescence has been widely used as a bioreporter in prokaryotic systems. The *lux* operon (*lux*CDABE) was originally isolated from *Vibrio fischeri* (Engebrecht et al., 1983), *Vibrio harveyi* (Cohn et al., 1983), and *Photorhabdus luminescens* (Szittner & Meighen, 1990)*.* The *lux* operon encodes for the luciferase enzyme (*luxAB*) and the long-chain aldehyde substrate (*luxCDE*) for that reaction. An assay employing bacterial bioluminescence does not require an external organic substrate; the only requirement is for oxygen (O2). A long chain aldehyde and a reduced flavin mononucleotide (FMNH2) are converted by luciferase (LuxAB) to a long chain carboxylic acid and FMN, producing light at 490 nm wavelength (Meighen & Dunlap, 1993). The *luxAB* (without *luxCDE*) can also be used as a bioreporter and while these strains also produce light at 490 nm, they are less suited for high

**1.1 Bioreporters** 

standard spectrophotometer.

biomarkers (Chalfie et al., 1994).

and expense to the assay.

throughput analysis due to additional handling steps (costly substrate addition) and additional cost.

The *luc* genes have been reported to be more sensitive than *lux*-based systems, however in a recent comparison of *luc*- and *lux*-based hormone-sensing bioreporters, Svobodova and Cajthaml (2010) determined that some *lux*-based bioreporters (BLYES/BLYAS bioassays, discussed below) are of comparable sensitivity and in some cases much more sensitive than *luc*-based bioreporters.

Several reviews are available on the properties and use of *luc*, *luxAB, luxCDABE, gfp,* and *gfp-*derived reporter genes in environmental systems (Hakkila et al., 2002; Keane et al., 2002; Ripp et al., 2010). Each of these reporter technologies has advantages and disadvantages depending on the application. For high throughput analysis of samples, bioreporters with the *luxCDABE* genes expressed are particularly well-suited for screening large numbers of samples. For both *luxAB*- and *lucFF*-based bioreporters, costly substrates must be continually added to the cells for visualization of the reaction. This increases not only handling difficulty but also costs to perform the assay. For GFP-based bioreporters, no exogenous substrates are necessary but fluorescent molecules must be excited by a light source to fluoresce. Each of these types of bioreporters produces signals for different lengths of time and has different light emission maxima and optimum temperatures. For example, while the *Photorhabdus luminescens* luciferase (Lux) is stable up to 42oC, firefly luciferase (Luc) has a temperature optimum at 25oC and is thermally inactivated above 30oC (Keane et al., 2002). Bioreporter fusions incorporating the full *lux* cassette are advantageous in that they do not require exogenous substrates, cell lysis is not required, the signal is quantitative and reproducible (King et al., 1990). Further, continuous on-line monitoring is possible (e.g. DiGrazia et al., 1991; Heitzer et al., 1994; Heitzer et al., 1992; King et al., 1990).

#### **1.2 Bacterial** *lux* **expression in** *Saccharomyces cerevisiae*

Prior to 2003, the *lux* genetic system was previously limited only to expression in prokaryotic systems. However, Gupta et al. (2003) were successful in expressing the *P. luminescens lux* cassette in the yeast *S. cerevisiae*. Specifically, the *luxA, -B, -C, -D*, and -*E* genes from *P. luminescens* and the *frp* gene from *Vibrio harveyi* were re-engineered for expression in *Saccharomyces cerevisiae*. The *lux* operon was engineered using two pBEVY yeast expression vectors (Miller et al., 1998), which allowed bidirectional, constitutive expression of the individual *luxA, -B, -C, -D*, and -*E* genes. The *luxA* and *luxB* genes were independently expressed from divergent yeast constitutive promoters GPD and ADH1 on pBEVY-U (Figure 1). The *luxCD* and *luxE-frp* genes were independently expressed from a second plasmid (pBEVY-L), also using the GPD and ADH1 promoters. An internal ribosome entry site (IRES) was inserted between the *luxC* and *luxD* genes and the *luxE* and *frp* genes. The IRES allows translation of multiple genes from a single promoter in eukaryotes (Hellen & Sarnow, 2001).

Constitutive expression of the *luxCDABEfrp* genes in *S. cerevisiae* W303a generated approximately 9,000,000 photons per second per unit optical density (Gupta et al., 2003). This is comparable to similar expression in prokaryotic systems. This was a significant milestone in expression of bacterial operons in lower eukaryotic systems and created possibilities for screening organic wastewater contaminants with mammalian health significance.

Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters 7

fluorescent protein (Bovee et al., 2007; Bovee et al., 2004) or the firefly luciferase bioreporter

While the YES and YAS assays were highly specific for their target compounds, the colorimetric assays have disadvantages including addition of the chromophore for color development and a 3-5 day reaction time. This latter requirement hindered their ability for high-throughput analysis. Further, after 3 -5 days of incubation, it was unknown if any oxidation reactions were occurring that may activate the target compound. Some newer colorimetric assays have dramatically shortened the time required for color development (4- 6 h) through the use of alternative substrates but have the disadvantage of requiring cell

To overcome these limitations, bioluminescent version of the YES and YAS reporters were developed by modifying the plasmid constructs of Gupta et al. (2003). Triple repeats of the human ERE were inserted in between the GPD and ADH1 constitutive promoters regulating the *luxA* and *luxB* genes, respectively (Figure 2) generating strain BLYES (Sanseverino et al., 2005). A similar strategy was used for strain BLYAS (Eldridge et al., 2007), which functions in the same way except that it contains the human androgen receptor gene on its genome and *luxAB* are under control of four androgen response elements (AREs), while the constitutive strain (BLYR) has both the *luxAB* and *luxCDEfrp* genes constitutively produced therefore it makes light constantly. The BLYR strain is used to determine whether samples or chemicals are toxic to the yeast, preventing false negatives. If a chemical is highly toxic, killing or inhibiting the cells, no light will be produced and it would be easy to mistake toxicity for no estrogenic response. However, if bioluminescence of the BLYR strain is reduced, since it produces light constitutively, it is obvious that toxicity exists in the sample.

Fig. 2. Schematic representation of *S. cerevisiae* BLYES. Estrogenic compounds cross the cell membrane and bind to the human estrogen receptor (hER). This complex interacts with estrogen response elements (RE) initiating transcription of *luxA* and *luxB*. *S. cerevisiae* BLYES contains the human estrogen receptor in its genome, while *S. cerevisiae* BLYAS has the

human androgen receptor in the genome.

(Bovee et al., 2004; Leskinen et al., 2005; Michelini et al., 2005).

lysis steps (Jaio et al., 2008).

Fig. 1. Schematic representation of *S. cerevisiae* BLYEV (currently known as BLYR). This strain produces light continuously by constitutive expression of the *luxCDABE* genes from *Photorhabdus luminescens* and the *frp* gene from *Vibrio harveyi*.
