Use of Saccharomyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds

Abstract

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17beta-estradiol over the concentration range of 1.2 x 10(-8) through 5.6 x 10(-12) M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 +/- 1.1) x 10(-10) and (2.4 +/- 1.0) x 10(-10) M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 x 10(-11) to 2.8 x 10(-9) M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.

FIG. 1.

Schematic representation of S. cerevisiae BLYES. Estrogenic compounds cross the cell membrane and bind to the estrogen receptor. This complex interacts with the ERE, initiating transcription of luxA and luxB. The gene for the human estrogen receptor is located on the chromosome.

FIG. 2.

PCR and cloning strategy for generating pUTK407. Sequential PCR steps were used to generate GPDEREADH. This construct was cloned into the BamHI-SpeI site of pUTK401 to generate pUTK407. Restriction site abbreviations: A, AvrII; B, BamHI; E, EcoRI; K, KpnI; N, NotI; P, PstI; Sc, SacI; Sl, SalI; Sp, SpeI; X, XmaI. Figure is not drawn to scale.

FIG. 3.

Three-dimensional plot of bioluminescence versus time for 17β-estradiol. Initial bioluminescence was observed in as little as 60 min for 2.8 × 10⁻⁹ M and reached a maximum at approximately 360 min. CPS, counts per second.

FIG. 4.

EC₅₀ dose-response profile of 17β-estradiol using S. cerevisiae BLYES (open circles) and S. cerevisiae hER (closed circles) reporter strains. The lower and upper limits of detection for each assay with 17β-estradiol were approximately 4.5 × 10⁻¹¹ and 2.8 × 10⁻⁹ M, respectively.

FIG. 5.

Linear regression analysis of EC₅₀ values determined by the bioluminescence and colorimetric assays. EC₅₀ values are listed in Table 3. A coefficient of determination of 0.99 was achieved.