The genomic evaluation of estrogen degradation by Rhodococcus equi ATCC 13557

Abstract

Estrogens estrone (E1), 17β-Estradiol (E2), and estriol (E3) are steroidal hormones produced naturally, and the synthetic estrogen 17α-Ethinylestradiol (EE2) is contained in the contraceptive pill. In 2012, the European Commission proposed international environmental limits in freshwater bodies on EE2 and E2, with Environmental Quality Standard (EQS) values of 0.035 and 0.4 ng/L, respectively. Despite their low concentrations (ng/L) in the environment, several studies demonstrated their harmful effect to aquatic organisms. Some bacteria have been identified to biodegrade estrogen with varied efficiency. Biodegradation is a low cost and sustainable method that could help in the removal of estrogens. This study aims to provide the framework with which to elucidate, identify and characterise those genes involved in estrogen degradation by a genomic evaluation of steroid degrading bacteria. Firstly, a putative estrogen biodegradation pathway was postulated, and a database of the major genes potentially involved in it was compiled, by combining existing information on the degradation of estrogen, and its better characterised analogue – testosterone, for which there is a complete degradation pathway. The genome of Rhodococcus equi ATCC13557, a known estrogen degrading bacterium, was sequenced, assembled and mapped. Genome analysis revealed a gene cluster potentially coding for enzymes involved in estrogen degradation; 3- carboxyethylcatechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase, 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, 3-(3-hydroxyphenyl)propionate hydroxylase, 3-oxosteroid 1-dehydrogenase and transcriptional regulator IcIR family. Specific primer sets were developed to target these genes, and using quantitative reverse transcription PCR (RT-qPCR), gene expression when exposed to E2 and EE2, was quantified relative to the control, without estrogen, and normalised to the reference genes, malate dehydrogenase and arginine deiminase. Gene expression of putative cytochrome P450 monooxygenase was upregulated initially in the presence of estrogen, therefore may be involved in one of the first steps in estrogen degradation. The RT-qPCR experiment was complemented by biodegradation studies, however, metabolites analysis is required to confirm the postulated estrogen biodegradation pathway.