性類固醇的微生物降解: 由模式物種延伸至環境樣本

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(1)Department of Life Science, National Taiwan Normal University Taiwan International Graduate Program on Biodiversity, Academia Sinica. Doctoral Dissertation :. Microbial Degradation of Sex Steroid Hormones: From Model Organisms to the Environmental Samples. Student: Yi-Lung Chen, PhD. Advisor: Yin-Ru Chiang, PhD. 106 5 May 2017.

(2) Summary Sex steroid hormones (SHs), a major group of endocrine disrupting agents, are often detected in aquatic environments. The most concerned SHs include estrogens (e.g., 17β-estradiol and estrone) and androgens (e.g., testosterone). Among the proposed remediation strategies, bacterial degradation has been considered an efficient and eco-friendly strategy for removing the SHs from the contaminated ecosystems. In this dissertation, I aimed to investigate the metabolic and phylogenetic diversity related to bacterial degradation of SHs from model organisms to the environemnt. By using culturable bacterial strains as model organisms, I demonstrated that strictly aerobic Sphingomonas sp. strain KC8 degrade estrogens through the 4,5-seco pathway; the essential meta-cleavage dioxygenase was isolated and characterized. Furthermore, through the genomic and transcriptomic analyses, I identified the catabolic gene clusters in the 4,5seco pathway of strain KC8, and in the 2,3-seco pathway for androgen biodegradation of Steroidobacter denitrificans DSM 18526. The omics studies on the model organisms enabled the environmental investigations of steroid biodegradation, for which I used the following approaches: (i) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identification of signature metabolites, (ii) identification of main catabolic players through next-generation sequencing techniques, and (iii) PCR-based identification of functional genes. This study is the. I.

(3) first integrated ‘omics’ investigation on the biochemical mechanisms and phylogenetic diversity of steroid biodegradation in the environment. In brief, Introduction provides the background information of SHs, current knowledge on their biodegradation, and my research objectives. The studies of androgen degradation: the genome of the androgen anaerobic decomposer, Steroidobacter denitrificans was completely sequenced and annotated. Transcriptomic data revealed the gene clusters that were distinctly expressed during anaerobic growth on testosterone; besides,. I. identified. the. bifunctional. 1-testosterone. hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, suggested as biomarkers to investigate androgen biodegradation. Based on the available biomarkers of androgen degradation, I investigated the biochemical mechanisms and corresponding microorganisms of androgen degradation in the anaerobic and aerobic sewage. Sewage samples collected from the Dihua Sewage Treatment Plant (Taipei, Taiwan) were incubated with testosterone (1 mM) anaerobically or aerobically. Androgen metabolite analysis indicated that denitrifying bacteria in anaerobic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assay showed androgen degradation in anaerobic sewage by Thauera spp. (mainly. T.. terpenica). through II. the. action. of. 1-testosterone.

(4) hydratase/dehydrogenase. Moreover, the 2.3-seco pathway utilized by T. terpenica 58Eu (DSMZ 12139) was also confirmed. By contrast, bacteria in aerobic sewage degraded androgens via the oxygenase-dependent 9,10seco pathway, and the metagenomic analysis indicated the apparent enrichment of Comamonas spp. (mainly C. testosteroni) and Pseudomonas spp. in sewage incubated with testosterone. I used the degenerate primers derived from the meta-cleavage dioxygenase gene (tesB) of various proteobacteria to track this essential catabolic gene in the sewage. The amplified sequences showed the highest similarity (87–96%) to tesB of C. testosteroni. Using quantitative PCR, I detected a remarkable increase of the 16S rRNA and catabolic genes of C. testosteroni in the testosteronetreated sewage. The studies of estrogen degradation: Using a tiered functional genomics approach, I deciphered the catabolic enzymes and genes involved. in. estrogen. biodegradation. by. a. wastewater. isolate,. Sphingomonas sp. strain KC8. I identified the initial intermediates of this catabolic pathway, including 4-hydroxyestrone, a meta-cleavage product, and pyridinestrone acid. The yeast-based estrogen assay suggested that pyridinestrone acid exhibits negligible estrogenic activity. Further genomic and transcriptomic analyses revealed that two gene clusters are specifically expressed in strain KC8 cells grown on 17β-estradiol. I also characterized 17β-estradiol dehydrogenase and 4-hydroxyestrone 4,5-dioxygenase responsible for the 17-dehydrogenation and meta-cleavage of the estrogen III.

(5) A-ring, respectively. The 4-hydroxyestrone 4,5-dioxygenase gene and the characteristic pyridinestrone acid were detected in two wastewater treatment plants and two suburban rivers in Taiwan. In conclusion, the catabolic genes and characteristic metabolites can be used as the biomarkers to investigate fate and biodegradation potential of estrogens in the environment.. Keywords:. 13. C-metabolomics, androgen, biodegradation, Comamonas,. denitrifying bacteria, ecophysiology, estrogen, extradiol dioxygenase, functional genomics, Illumina MiSeq, molybdoenzyme, RNA-Seq, Sphingomonas, steroid hormones, Steroidobacter, sewage treatment plant, Thauera. IV.

(6) Acknowledgement This dissertation would not have been possible without the help and the guidance of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. First and foremost, I offer my sincerest gratitude to my supervisor, Yin-Ru Chiang (. ), who has supported me throughout my. dissertation with his patience and knowledge. Without him, this dissertation would not have been completed. One simply could not wish for a better or friendlier supervisor. Special thanks to the research assistants, Chia-Hsiang Wang ( ), Chia-Ying Yang (. ) and Fu-Chun Yang (. ). Without their. supports, I won’t be familiar with techniques for studies of steroid degradation in a short time. I gratefully acknowledge to Jiun-Yan Ding ( Jen (. ) and Shih Chao-. ). They never tired of answering my questions on molecular. methods and data analysis, especially at dinner. Many thanks go to Chang-Ping Yu (. ), providing me. Sphingomonas sp. strain KC8 and its genome sequences. Thanks to TzongHuei Lee (. ), providing his effort on structure identification of the. pyridinestrone acid. Thanks to King Siang Goh ( (. ) and Ying-Mi Lai. ) on protein characterization and metabolite quantification. respectively. It is thanks to Sen-Ling Tang ( V. ), Mei-yeh Lu (. ),.

(7) Ching-Hung Tseng ( (. ), Chen-Yu Yang (. ) and Chueh-Pai Lee. ) that I realize the power of Next Generation Sequencing and. scratch its surface. Lack of accurate and appropriate writing, my intellectual endeavor will not be easily understood. I gratefully appreciate Wael Ismail El Moslimany, Po Hsiang Wang (. ) and Jun Yao Chen (. ) for. their careful reading of the manuscript and helpful comments and suggestions. Furthermore, I appreciate the finical support from the TIGP. All people in the TIGP BIODIV (. ) are my strong. backup. That is a long list included. and. , et cetera.. Finally, I would also like to thank my parents, Kuei-Hsiang Chen (. and Li-Hua Huang. ). They always support and. encourage me with their best wishes.. Yi-Lung Chen (. ) / r90241213@gmail.com Nankang, Taiwan May 7th 2017. VI.

(8) Curriculum Vitae Researchgate: https://www.researchgate.net/profile/Yi-Lung_Chen. EDUCATION. 2012-2017 2010-2012 2006/7 2001-2006. 1994-1998 WORK 2006-2010 EXPERIENCE 2002-2003 1998-2000. CRUISE. Biodiversity program, Taiwan International Graduate Program Master degree Erasmus Mundus MSC in Marine Biodiversity and Conservation, Germany and France Virus Ecology Workshop in Plymouth, UK Master degree Division of Marine Biology & Fisheries, Institute of Oceanography, National Taiwan University, Taiwan Qualified secondary Educational Program for Secondary school teacher School Teachers, Center for Teacher Education of National Taiwan University, Taiwan Bachelor degree Department of Botany, National Chung Hsing University, Taiwan Research assistant Research Center for Environmental Changes, Academia Sinica, Taiwan Substitute teacher Taipei JingMei Girls High School, Taiwan Military service Army 101st Amphibious Reconnaissance Battalion, Taiwan ( ). Part-Time 2003/5-7. Technical assistant. 2001/4-7. Research assistant. 1996-1997. Library management & activities advisory R/V Kilo Moana, USA West Pacific Ocean Ocean Research No.1, East China Sea, five cruises Taiwan Ocean Research No.2, East-North part of Taiwan, Asia Taiwan sandstorm project, fifteen cruises Weekly or biweekly sampling in the Fei-Tsui reservoir, Taiwan Best award of poster 8th Asian Symposium on Microbial presentation Ecology, Taipei, Taiwan Travel grant International Symposium on Microbial Ecology (ISME), Montreal, Canada Best award of poster Cross-Strait Symposium of presentation Environmental microbiology, Taichung, Taiwan Best award of poster Symposium of Frontier of Microbial presentation Ecology, Taipei, Taiwan. 2009/3-4 2004-2009 2004-2009. AWARD. Ph.D.. 2003-2010 2016/10/1-2 2016/8 2015/11/6-8 2014/9/4-5. VII. Education and Technology Group, National Taiwan University Digital Museum Project, National Museum of Natural Science, Taiwan Wild Bird Association of Taiwan.

(9) Contributions and Publications This Ph.D. thesis is based on a collection of several manuscripts. At the time of submission, two manuscripts were published, and the third was accepted in refereed journal. These manuscripts, as listed below. They have been slightly modified for consistency in formatting. In all manuscripts, I am the first or co-first author and my supervisor Dr. Yin-Ru Ching is the corresponding author. The content of each manuscript is dominated by my intellectual effort although there has been a contribution by several other individuals who are recognized as co-authors. The roles of the co-authors are explained in detail below.. [1]! Yi-Lung Chen, Chia-Hsiang Wang, Fu-Chun Yang, Wael Ismail, PoHsiang Wang, Chao-Jen Shih, Yu-Ching Wu, and Yin-Ru Chiang. (2016) Identification of Comamonas testosterone as an active player in aerobic androgen biodegradation in sewage, Scientific Reports, 6, 35386. [2]! Fu-Chun Yang, Yi-Lung Chen, Sen-Lin Tang, Chang-Ping Yu, PoHsiang Wang, Wael Ismail, Chia-Hsiang Wang, Jiun-Yan Ding, Cheng-Yu Yang, Chia-Ying Yang, and Yin-Ru Chiang. (2016) Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment. ISME J. doi: 10.1038/ismej.2015.255 (2016).. VIII.

(10) [3]! Yi-Lung Chen, Chang-Ping Yu, Tzong-Huei Lee, King-Siang Goh, Kung-Hui Chu, Po-Hsiang Wang, Wael Ismail, Chao-Jen Shih, and Yin-Ru Chiang. (2017) Bacterial estrogen degradation proceeds through the extradiol dioxygenase-mediated 4,5-seco pathway. Cell Chemical Biology. Accepted. The ideas in the first manuscript [1] were conceived by myself, and I carried out the work, including the design of the experiment, methodology, analysis. Dr. Yin-Ru Chiang and I were involved in the manuscript writing and revision. Chia-Hsiang Wang, Fu-Chun Yang and Yu-Ching Wu developed the method for the processing the samples. Dr. Chao-Jen Shih conducted a PCR experiment. Po-Hsiang Wang, Dr. Wael Ismail and Dr. Yin-Ru Chiang actively participated in the discussion of the experiment, results, and reviewing of the manuscript. In manuscript [2], Fu-Chun Yang and I conceived the key ideas, conducted the experiments, Dr. Yin-Ru Chiang and I wrote the manuscript and performed the revisions. Cheng-Yu Yang, Dr. Jiun-Yan Ding and Dr. Sen-Lin Tang assisted with conducting genome analysis with COG, and the RNA-Seq analysis. Fu-Chun Yang and Chia-Hsiang Wang conducted the most enzyme experiments, included cloning and over-expressing the bifunctional 1-testosterone hydratase/dehydrogenase. Po-Hsiang Wang, Dr. Wael Ismail and Dr. Yin-Ru Chiang actively participated in the discussion of the experiment, results, and reviewing of the manuscript. IX.

(11) In manuscript [3], I initiated the research topic, conducted the experiment, sample and data analysis. I and Dr. Yin-Ru Chiang wrote and revised the manuscript. Dr. Chang-Ping Yu and Dr. Kung-Hui Chu provided two model organisms for estrogen degradation experiments and the tricky of incubation. Dr. Tzong-Huei Lee analyzed the MNR data to resolve and identify the structure of metabolites. Cheng-Yu Yang assisted with conducting resting cell experiment. Dr. King-Siang Goh and I conducted the most enzyme experiments, included cloning and overexpressing the dehydrogenase; enzyme active, subtracts preference and cofactors analysis. Po-Hsiang Wang, Dr. Chao-Jen Shih, Dr. Wael Ismail and Dr. Yin-Ru Chiang actively participated in the discussion of the experiment, results, and reviewing of the manuscript.. X.

(12) Table of Contents Summary…………………………………………………….. I. Acknowledgement………………………………………….....V Curriculum Vitae………………………….………………...VII Contributions and Publications…………………………. VIII Table of Contents……………………………………………. XI List of Tables……………………………………………….XVII List of Figures……………………………………………….XIX List of Appendix Tables………………………………...…XXIV List of Appendix Figures……………………………….....XXVI List of Abbreviations………………………………….…XXVII 1 Introduction 1.1! Sex. steroid. hormones:. structure,. properties,. occurrence, and biological significance……………….1 1.2! Sex steroid hormones: environmental impact………...3 1.3! Sex. steroid. hormones:. biotransformation,. biodegradation, and mineralization…………………...5 1.3.1! Microbial androgen degradation……………………….6 1.3.2! Microbial estrogen degradation………………………...9 1.3.3! Microbial progestogen degradation……………………12. 1.4! Research objectives…………………………...………12 2! Materials and Methods 2.1! Chemicals and bacterial strains…………………….17 XI.

(13) 2.1.1! Chemicals……………………………………….….17 2.1.2! Bacterial strains and plasmids……….………………….17. 2.2! Bacterial. cultures……….…………….……………….17. 2.2.1! Growth of Steroidobacter denitrificans DSM 18526…….17 2.2.2! Growth of Sphingomonas sp. strain KC8……………....18 2.2.3! Other bacteria……….…………….………………….20. 2.3! Preparation of cell extracts……………………...…....20 2.4! Determination of activity of sex steroid hormones….21 2.4.1! lacZ-based yeast androgen bioassay………………....21 2.4.2! lacZ-based yeast estrogen bioassay…………………....22. 2.5! Purification. and. characterization. of. steroid-. transforming enzymes………………………...……....23 2.5.1! Purification of 4-hydroxyestrone 4,5-dioxygenase (OecC) from estrone-grown Sphingomonas sp. strain KC8…….23 2.5.2! SDS-PAGE, MS/MS analysis, and protein identification.25. 2.6! Genome sequencing, assembly and annotation……...25 2.6.1! Steroidobacter denitrificans genome………………....25 2.6.2! Sphingomonas sp. strain KC8 genome………………...26. 2.7! RNA extraction and transcriptomic analysis………...27 2.7.1! RNA-Seq sequencing of Steroidobacter denitrificans mRNA…………………………………………….….…..27 2.7.2! RNA-Seq sequencing of Sphingomonas sp. strain KC8 mRNA………………………………………….….…......28 XII.

(14) 2.8! Sampling sites and sample preparation……….……….29 2.8.1! Sampling sites…………………………………………….29 2.8.2! Sample collection and preparation……….…………...….31. 2.9! Bacterial community analysis……….…………………...33 2.9.1! Illumina MiSeq sequencing of bacterial 16S rRNA amplicons….…….………………………………….…... 33 2.9.2! 16S rRNA gene-based taxonomic analysis……….……...35. 2.10!Analytical chemical methods…………………….……...35 2.10.1!Nucleic acid analysis…………………….……………...35 2.10.2!Determination of total protein concentration……….….35 2.10.3!Thin layer chromatography (TLC) ……….…………….36 2.10.4!High performance liquid chromatography (HPLC) …….36 2.10.5!Mass spectrometry (MS) ……….…………………...….37. 2.11!Molecular biological methods……….………………….39 2.11.1!General DNA manipulations……….………………….39 2.11.2!Polymerase chain reaction (PCR) ……….……….…….39 2.11.3!Quantitative real-time PCR (qPCR) ……….…………....41 2.11.4!Gene cloning and sequencing……….……….………....42 2.11.5!Phylogentic analysis of DNA and protein sequences…....42 2.11.6!Clone, over-expression, purification and properties of recombinant protein, OecChis……….……….……….......43. 3! Results 3.1! Omics studies on the model organisms……………...….48 XIII.

(15) 3.1.1! Steroidobacter denitrificans genome…………….…...….48 3.1.2! Comparative. transcriptomics. of. Steroidobacter. denitrificans…………………………………………...….50 3.1.3! Phylogenetic analyses of essential catabolic enzyme involved in anaerobic androgen degradation pathway……54 3.1.4! Sphingomonas sp. strain KC8 degrades natural estrogens through the steroid 4,5-seco pathway………………….…55 3.1.5! Genome of Sphingomonas sp. strain KC8……………...…56 3.1.6! Comparative transcriptomics of Sphingomonas sp. strain KC8……………............................................................…58 3.1.7! Functional confirmation of oecA……………………....…59 3.1.8! Purification and characterization of OecC…………….…60. 3.2! Microbial steroid catabolism in the environmental samples……………..............................................................…63 3.2.1! Identification of androgen metabolites in the sewage….…63 3.2.2! Phylogenetic identification of androgen-degrading bacteria in the testosterone-spiked sewage……………………...…65 3.2.3! PCR amplification of the functional genes for androgen degradation in the sewage……………………...............…67 3.2.4! Quantitative PCR confirmed the remarkable increase of the 16S rRNA and catabolic genes of Comamonas testosteroni in the testosterone-spiked sewage……………………...…70 3.2.5! Prevalence of pyridinestrone acid in aquatic ecosystems XIV.

(16) during estrogen biodegradation……………………......…71 3.2.6! Presence and diversity of 4-hydroxyestrone 4,5-dioxygenase genes in the activated sludge……………………...........…72. 4! Discussion 4.1! Catabolic genes involved in androgen degradation by Steroidobacter denitrificans……………………..............…74 4.2! atcA. gene. as. a. biomarker. for. environmental. investigation of anoxic androgen biodegradation….…77 4.3! Biochemical mechanisms and catabolic enzymes involved in estrogen degradation by Sphingomonas sp. strain KC8……………………............................................…78 4.4! Catabolic strategy of Sphingomonas sp. strain KC8 towards androgens and estrogens…………………….…81 4.5! Other microorganisms potentially degrade estrogens through the 4,5-seco pathway………………………….…82 4.6! Pyridinestrone acid as a biomarker for environmental investigation of estrogen biodegradation…………….…84 4.7! Degradation of sex steroid hormones on microbe-host interactions ……………………..........................................…85 4.8! General feature of the bacterial communities and androgen transformation in the sewage of DHSTP…86 4.9! Proposed androgen degradation pathways and the primary degraders in the testosterone-spiked sewage XV.

(17) sample……………………....................................................…88 4.9.1! The anaerobic androgen degradation pathway function in the aerobic sewage……………………...................................88 4.9.2! Thauera spp. may be the primary androgen degraders in the anoxic sewage………………….…...................................89 4.9.3! The aerobic androgen degradation pathway function in the oxic sewage……………………........................................90 4.9.4! Comamonas teostosteroni was identified as an androgen degrader in the aerobic sewage………………………...…91. 4.10!Limitation of 16S rRNA amplicon analysis on inferring metabolic ability………………………...........…95 4.11!The aerobic estrogen degradation pathway functions in aquatic ecosystems……………………………...........…96 4.12!Sequence diversity of the functional gene for aerobic estrogen degradation in the sewage…………………...…97 4.13!Comparison with other culture-independent methods on in situ microbial degradation of estrogens………….98 4.14!Future prospects on in situ techniques for studying microbial. degradation. of. sex. steroid. hormones……………………………………………...……....99 5! Reference…………………….......................................................102 6! Appendix……………………........................................................205. XVI.

(18) List of Tables Table 1.!. List of bacteria capable of completely degrading sex steroid hormones.……………………..........................................131. Table 2.!. Advantages. and. disadvantages. of culture-independent. methods applied to study functional ability and taxonomic identity of sex-steroid-hormones...……………….…….136 Table 3.!. Bacterial strains and plasmids used in this study...……….138. Table 4.!. The chemical components involved in the chemical defined medium. for. denitrifying. growth. of. Steroidobacter. denitrificans...……………….………………………...….139 Table 5.!. The chemical components involved in the chemical defined medium. for. aerobic. growth. of. Steroidobacter. denitrificans………………………………………………141 Table 6.!. The ingredient of R2A medium...………….……….…….143. Table 7.!. The ingredient of nutrient agar...………….……….…….143. Table 8.!. The ingredient of trypticase soy broth agar...……….…….143. Table 9.!. General physical and chemical parameters of the sampling sites……………………………………………….………144. Table 10.! Primers used in this study...………….……….………….145 Table 11.! Expression profile of the Steroidobacter denitrificans genes which are probably involved in anaerobic testosterone degradation...…………..……………………...………….146 XVII.

(19) Table 12.! Molybdopterin-containing subunits of bacterial proteins used for constructing the phylogenetic tree in Figure 11...…….148 Table 13.! A selection of the extradiol dioxygenases used for constructing the phylogenetic tree in Figure 22 and 42.......149 Table 14.! 3,4-DHSA. dioxygenases. used. for. constructing. the. phylogenetic tree shown in Figure 36...………………….151 Table 15.! Putative oecB and oecC genes found in the genomes of 12 bacterial strains...………….………………….………….152. XVIII.

(20) List of Figures Figure 1.! Common sex steroid hormones...………………………….153 Figure 2.! Proposed catabolic pathways of testosterone...………...….154 Figure 3.! Comparison of the intermediates involved in anaerobic testosterone. catabolic. pathway. (demonstrated. in. Steroidobacter denitrificans DSMZ 18526) with those involved in anaerobic biodegradation of cyclohexanol (demonstrated 14773) Figure 4.! Proposed. in. Alicycliphilus. denitrificans. DSMZ. …………………………………………………...155 bacterial. degradation. pathways. of. natural. estrogens) ………………………….……………………...156 Figure 5.! Locations for collecting the environmental samples...…….157 Figure 6.! Conserved regions in the amino acid sequences of AtcA-like proteins and the deduction of the atcA gene probe. ...…...….158 Figure 7.! Multiple alignment of amino acid sequences of 3,4-DHSA dioxygenase (TesB) showing conserved regions in the TesB proteins that were used to design a tesB-specific gene probe...……………………………………..………………159 Figure 8.! Conserved regions in the amino acid sequences of OecC-like proteins and the deduction of the oecC gene probe...…...…160 Figure 9.! Genome atlas of Steroidobacter denitrificans and the gene clusters for steroid degradation...……………………….…161. XIX.

(21) Figure 10.! Global gene expression profiles (RNA-Seq) of Steroidobacter denitrificans grown under different conditions...……….…163 Figure 11.! Phylogenetic tree of the members of the xanthine oxidase family based on the amino acid sequences of molybdopterincontaining subunits...…………………………...……….…164 Figure 12.! UPLC–ESI–HRMS analysis indicated substrate consumption (A) and sequential production of 4-hydroxyestrone (B) and pyridinestrone acid (C) by estrone-grown strain KC8 cells………………………………………………………...165 Figure 13.! Proposed estrogen aerobic biodegradation pathway of Sphingomonas sp. strain KC8, and the gene clusters together with. corresponed. expression. under. different. steroid. enrichments…………………………………………….….166 Figure 14.! Estrogenic activities of different catabolic metabolites of estrogen, and the cell suspensions of strain KC8 incubated with estrone………………………………………………….….168 Figure 15.! Androgen aerobic biodegradation pathway of Sphingomonas sp. strain KC8, and the gene cluster together with corresponed expression under different steroid enrichments...……….…169 Figure 16.! Purification. of. 4-hydroxyestrone. 4,5-dioxygenase. (OecC)………………………………………………….….170 Figure 17.! ESI–MS (A) and UV/Vis absorption (B) spectra of the HPLCpurified ring-cleaved product...…………………...…….…171 XX.

(22) Figure 18.! ESI–MS analysis indicating that OecChis requires O2 for the transformation of 4-hydroxyestrone to the ring-cleaved product………………………………………………….….172 Figure 19.! Transformation of the ring-cleaved product to pyridinestrone acid requires a nitrogen donor…....…………………...……173 Figure 20.! pH effect and enzyme kinetics of OecChis...………………174 Figure 21.! Substrate. preference. of. OecChis. determined. spectrophotometrically by following the appearance of ringcleaved products.………….…....…………………...……175 Figure 22.! Phylogenetic tree of selected extradiol dioxygenases…….176 Figure 23.! UPLC-APCI-MS/MS analysis of the ethyl acetate extracts of the anoxic DHSTP sewage treatments………………….….177 Figure 24.! Time course of androgenic activities in the negative control (autoclaved sewage incubated with testosterone and nitrate) and three treatments of anoxic DHSTP sewage…………….179 Figure 25.! UPLC-APCI-MS/MS analysis of the ethyl acetate extracts of anoxic. DHSTP. sewage. incubated. under. different. conditions………………………………………………….180 Figure 26.! The extracted ion chromatograms (m/z = 305.21 for 2,3SAOA) revealed the presence of the signature metabolite in DHSTP sewage incubated with testosterone……………….181 Figure 27.! UPLC-APCI-MS/MS analysis of the ethyl acetate extracts of the DHSTP sewage treatment samples……………….…….182 XXI.

(23) Figure 28.! UPLC-APCI-MS/MS analyses of the ethyl acetate extracts of testosterone-amended sewage Replicate 2……...………….184 Figure 29.! Androgenic activities of different compounds and sewage sample………………….….……………………………….185 Figure 30.! Genus-level phylogenetic analysis (Illumina MiSeq) revealed the temporal changes of the bacterial community structures in the anoxic and aerobic DHSTP swage incubated under different conditions…………………...….………………………….186 Figure 31.! Temporal variation of Thauera spp., atcA gene and its diversity in the testosterone-treated sewage……….……….188 Figure 32.! Genus-level phylogenetic analysis (IlluminaMiSeq) revealed the temporal changes in the bacterial community structures in various aerobic sewage treatment samples………...……….190 Figure 33.! The pie chart represents the relative abundances of individual species in the genus of Pseudomonas (100%) in the aerobic DHSTP sewage incubated with testosterone (1 mM) for 96 h……………………...…………………………………191 Figure 34.! Temporal changes in the bacterial community structures in sewage Replicate 2……………………………….……….192 Figure 35.! The anaerobic degradation of testosterone by Thauera terpenica 58Eu…………………………...……….……….193 Figure 36.! Phylogenetic tree of 3,4-DHSA dioxygenases involved in the meta-cleavage of the steroidal A-ring………...……...…….194 XXII.

(24) Figure 37.! PCR-based functional assay with degenerate primers derived from the proteobacterial tesB genes………...……….…….195 Figure 38.! PCR-based functional assay using the DNA extracted from testosterone-amended sewage Replicate 2 as the templates.197 Figure 39.! Quantitative real-time PCR indicated the temporal changes in the Comamonas testosteroni 16S rRNA (A) and tesB (B) gene copies in the testosterone-treated sewage………...…….….199 Figure 40.! Detection of molecular markers of the 4,5-seco pathway in 0.33 µM [3,4C-13C]estrone-treated activated sludge collected from. two. wastewater. treatment. plants. DHSTP. and. LKCSTP…………………………………………………...200 Figure 41.! Temporal changes in estrone consumption and. 13. C-labeled. pyridinestrone acid (PEA) production in 0.33 µM [3,4C13. C]estrone-treated water collected from the Tamsui River. (N1~N5) and Gaoping River (S1~S3) ………...………..….201 Figure 42.! The phylogenetic tree of deduced amino acid sequences of oecC fragments obtained from the activated sludge of DHSTP and LKCSTP………………………….…………………...202 Figure 43.! Thin-layer chromatograms (TLC) showing the transformation of testosterone to androst-4-en-3,17-dione by the aerobically grown Comamonas composti cells………...…………....….204. XXIII.

(25) List of Appendix Tables Table A1.!Twenty tesB DNA fragments obtained from the aerobic DHSTP sewage (incubated with testosterone for 48 h ; Replicate 1) by using tesB-specific degenerate primers tesBf1/tesB-r1………...……………………………..…....….205 Table A2.!Twenty tesB DNA fragments obtained from another testosterone-amended sewage replicate (incubated with testosterone for 48 h ; Replicate 2) by using the degenerate primers tesB-f1/tesB-r1………...………...……………209 Table A3.!Annotation of Steroidobacter denitrificans genome…….213 Table A4.!Comparative. transcriptomics. of. Steroidobacter. denitrificans………………………………………….….213 Table A5.!1H (600 MHz)- and. 13. C (150 MHz)-NMR spectral data of. pyridinestrone acid (Compound 1) and 4-hydroxyestrone (Compound 2) ……...………...…………………………214 Table A6.!Genome annotation and comparative transcriptomics of Sphingomonas sp. strain KC8……...………...…………214 Table A7.!Twenty atcA sequences amplified from anoxic DHSTP sewage (incubated with testosterone and nitrate for 120 h) by using atcA-specific primers AtcA-f1/AtcA-r1……...…215. XXIV.

(26) Table A8.!The individual sequences of 40 oecC fragments obtained from the activated sludge of DHSTP and LKCSTP using a pair of oecC-specific degenerate primers oecC-f1/oecCr1……………………………………………………...222. XXV.

(27) List of Appendix Figures Figure A1.! Purification. and. characterization. of. 1-testosterone. hydratase/dehydrogenase (AtcABC) from Steroidobacter denitrificans grown anaerobically on testosterone…....232 Figure A2.! Two-dimensional NMR spectra [COSY (A), HSQC (B), and HMBC (C)] of HPLC-purified pyridinestrone acid…………………………………………………...234 Figure A3.! Functional characterization of recombinant 17β-estradiol dehydrogenase (OecAhis) ……………………….........236 Figure A4.! Quantitative real-time PCR standard curves obtained using primer pairs Eub (circles), CteA2 (triangles) and TesBq (squares) using 4-fold serial dilutions of Comamonas testosteroni ATCC 11996 genomic DNA as template DNA………………………...........................237 Figure A5.! UPLC–ESI–MS/MS. identification. of. 13. C-labeled. pyridinestrone acid in [3,4C-13C]estrone-treated water collected from Five sampling sites (N1~N5) along the Tamsui River………………………............................238 Figure A6.! UPLC–ESI–MS/MS. identification. of. 13. C-labeled. pyridinestrone acid in [3,4C-13C]estrone-treated water collected from Three sampling sites (S1~S3) along the Gaoping River………………………..........................239. XXVI.

(28) List of Abbreviations 2,3-SAOA 3,4-DHSA 3-HSA 4-OH-E1 A A/O AD ADD Ali. Alt. APCI ATCC Azo. bp C. ca. cDNA CDS Chol CPRG Da DCPIP DEAE DHSTP DMSO DNA DOC DSMZ dT E. E0 E1 E2. 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid 3,4-dihydroxy-9,10-seco-androsta-1,3,5(10)triene-9,17-dione 3-hydroxy-9,10-seco-androsta-1,3,5(10)-triene9,17-dione 4-hydroxyestrone ampere anaerobic/oxic androst-4-en-3,17-dione androsta-1,4-diene-3,17-dione Alicycliphilus Altererythrobacter atmospheric pressure chemical ionization American Type Culture Collection Azoarcus base pair Comamonas circa complementary deoxyribonucleic acid coding DNA sequences cholesterol chlorophenol red-β-D-galactopyranoside dalton 2.6-dichlorophenolindophenol diethylaminoethyl Dihua Sewage Treatment Plant Dimethyl sulfoxide deoxyribonucleic acid Dissolved organic carbon Deutsche Sammlung von Mikroorganismen und Zellkulturen 1-dehydrotestosterone Escherichia 16-estratetraen-3-ol estrone 17β-estradiol XXVII.

(29) E3 EDTA EE2 EIC ESI FAD FISH FPLC g g h HEPES HPLC HRMS ICP IPMB IPTG l LB LKCSTP M M. m/z MALDI MAR min mRNA MS MS/MS mT NAD nanoSIMS NCBI NMR Nov. OD P.. estriol ethylenediaminetetraacetic acid ethinylestradiol extracted ion current electrospray ionization flavin adenine dinucleotide fluorescence in situ hybridization fast protein liquid chromatography gram gravity hour(s) 2-[4-(2-hydroxyethyl)piperazin-1yl]ethanesulfonic acid high performance liquid chromatography high resolution mass spectrometry inductively coupled plasma Institute of Plant and Microbial Biology isopropyl β-D-1-thiogalactopyranoside liter Luria-Bertani Liukuaicuo Sewage Treatment Plant molar Mycobacterium mass-to-charge ratio matrix-assisted laser desorption/ionization microautoradiography minute(s) messenger RNA mass spectrometry tandem mass spectrometry 17α–methyltestosterone nicotinamide adenine dinucleotide nanometer-scale secondary-ion mass spectrometry National Center for Biotechnology Information nuclear magnetic resonance Novosphingobium optical density Pseudomonas XXVIII.

(30) P4 PAGE PCR qPCR rDNA RNA RNA-Seq RPKM s S Sdo. SDS SHs SIP Stl. T. TIC TLC Tris UPLC UPLC–ESI–HRMS UV V v/v Vis. progesterone polyacrylamide gel electrophoresis polymerase chain reaction quantitative real-time PCR ribosomal DNA ribonucleic acid RNA sequencing reads per kilobase per million second simens Steroidobacter sodium dodecyl sulfate sex steroid hormones stable isotope probing Sterolibacterium Thauera total ion current thin layer chromatography tris(hydroxymethyl)aminomethane ultra-performance liquid chromatography ultra-performance liquid chromatographyelectrospray ionization-high resolution mass spectrometry ultrviolet voltage volume percentage visible. XXIX.

(31) 1 Introduction 1.1 Sex steroid hormones: structure, properties, occurrence, and biological significance Sex steroid hormones (SHs), including androgens, estrogens and progestogens, are naturally occurring compounds belonging to a class of terpenoid lipids. SHs are characterized by a flat and relatively rigid carbon skeleton composed of four fused alicyclic rings: three cyclohexane rings are designated A, B, and C, whereas the fourth ring, a cyclopentane, designated D, and the carbon atoms of SHs are numbered using a unique scheme (Figure 1). A remarkable property of SHs is their extremely low aqueous solubility. For example, the solubility of natural estrogens [17βestradiol (E2) and estrone (E1)] in neutral aqueous solutions is approximately 1.3-1.5 mgl-1 (Shareef et al., 2006) at room temperature, whereas the experimental aqueous solubility of testosterone at 25 ºC is approximately 23.4 mgl-1 (Barry and Eleini, 1976). SHs are not only identified in vertebrates but also some invertebrates such as insects (Mechoulam et al., 1984) and corals (Tarrant et al., 2003). Several pieces of evidence indicate that exogenous SHs-enriched invertebrates altered their growth and reproduction (Scott 2012, Tarrant et al., 2004). Moreover, progestogen could be a key to switch sexual/asexual production of a marine rotifer, Brachionus manjavacas (Stout et al., 2010). Thus far, no sufficient evidence reports that these invertebrates can de novo synthesize SHs. Using cholesterol as the precursor, vertebrates synthesize 1.

(32) steroid hormones, which are highly bioactive and regulate a variety of physiological processes including immunology, reproduction and homeostasis (Ferrier 2017). SHs are produced in the endocrine gonads and peripheral tissues and secreted into the circulation in which SHs are mostly bound to either specific globulin or non-specific albumin (Chen and Farese, 1999). When trafficking in target cells, SHs must free themselves from their blood-solubilizing proteins. SHs in free forms interact with target cells by either through a rapid, non-genomic mechanism or through a slow, genomic one: SHs can combine with membrane-embedded receptors and initiate intracellular signaling cascades via the non-genomic mechanism; alternatively, SHs can bind to the corresponded receptors to form complex structures in the cytosol. Once entering the cell nucleus, these complexes combine with the specific DNA fragments to control expressions of their target genes. No report indicate animals can completely mineralize SHs. Instead, in the liver, SHs undergo structural modifications, and are converted into inactive excretion products. The solubility of the resulting products is promoted through conjugation with glucuronic acid or sulfate. Approximately 20~30% of these conjugates are secreted into the bile and eventually, excreted in feces. The rest is excreted in urine after being filtered from blood plasma in the kidneys (Ferrier 2017, Hadd and Blickenstaff 1969) The daily amount of SHs excreted by humans and animals varies due to sex, physiological and development states, and health 2.

(33) condition. For example, daily estrogen excretion by a pregnant woman can be up to 7 mgd-1 (Johnson et al., 2000). As to farm animals, Lange et al. (2002) estimated that the annual estrogen excretion in the European Union and the United States could be up to 33 and 49 metric tons, respectively. In the environments, these conjugated SHs were deconjugated through microbial activities (Duong et al., 2011, Panter et al., 1999). 1.2 Sex steroid hormones: environmental impact SHs are ubiquitous in various environments such as manures, farmlands, soils, river, lake, estuarine sediments, ocean and even to the deep-sea sediments (Fernandez et al., 2017, Griffith et al., 2016, Moschet and Hollender 2009, Ying et al., 2002). Steroid hormones are discharged into the environment through various routes, including the effluents of sewage treatment plants and the agricultural application of livestock manure and municipal sewage biosolids as fertilizers (Hanselman et al., 2003, Lorenzen et al., 2004). Moreover, phytosterols in pulp and paper mill effluents were transformed to androgens by microorganisms in river sediments, consequently influencing the physiology and development of fishes (Jenkins et al., 2001, 2003). SHs are often detected at nanograms to micrograms per liter concentrations in surface water (Baronti et al., 2000, Chang et al., 2011, Chen et al., 2010, Fan et al., 2011, Kolodziej et al., 2003, Ternes et al., 1999, Yamamoto et al., 2006). Quantifying five classes of steroid hormones in the surface waters of Beijing (China) revealed that 3.

(34) androgens (up to 1.9 µg l-1) were the most abundant steroids in urban rivers (Chang et al., 2009). Since 1989, numerous documents have reported that exogenous SHs, including natural and synthetic ones, cause profound and adverse effects to aquatic life from different levels of biological organization. At DNA levels, it is reported that the genomic DNA of developing barnacle larvae (Elminius modestus) was damaged and mutated after exposed to E2 (Atienzar et al., 2002). At protein levels, both the cypris major protein and vitellogenin were produced when male individuals were exposed to E2 (Billinghurst et al., 2000), the indicators as the feminization of male animals. At individual levels, exogenous E2 led to the settlement inhibition of the crypts larva of the barnacle Balanus amphitrite (Billinghurst et al., 1998). In addition, an integrated laboratory and field study indicate that E2 delay molt development and downstream migration in Atlantic salmon (Salmo sala, Madsen et al., 2004). A report also revealed that coral colonies treated with E2 released fewer egg-sperm bundles and coral fragments had lower skeletal growth rates (Tarrant et al., 2004); By contrast, exogenous androgenic compounds induced the masculinization (e.g., anal ray elongation) of female fishes (Turner, 1960). At population levels, Schwindt et al. (2014) conducted a year-long study on three generations of fathead minnows and found that environmental estrogens disrupted fish population dynamics through direct and transgenerational effects on the survival and fecundity of individuals. In a 7-year, whole-lake experiment, Kidd et al. 4.

(35) (2007) found that a synthetic estrogen, ethinylestradiol (EE2), resulted in the collapse of a fathead minnow population. At community levels, a comprehensive evaluation indicated that an aquatic food web was disturbed through EE2 exposure (Kidd et al., 2014). Exogenous SHs are not only endocrine-disrupting agents but may also serve as potential carcinogens to human. Several reports have indicated that E2 can trigger breast cancer (Davis et al., 1993; Safe, 2004). The World Health Organization has classified estrogens as Group 1 carcinogens. Please refer to the website of International Agency for Research on Cancer (http://monographs.iarc.fr/ENG/Classification/latest_classif.php). Moreover, some catechol metabolites of estrogens (e.g., 4-hydroxyestrone) are also reportedly carcinogens (Fernandez et al., 2006; Liehr et al., 1986). 4-Hydroxyestrone and other catechol estrogens are present in animal urine (Emons et al., 1980).. 1.3 Sex steroid hormones: biotransformation, biodegradation, and mineralization To avoid the potential risk of estrogens, several countries have prohibited estrogens as additive agents in cosmetics. In addition, several remediation processes have been proposed and developed to remove these SHs from the environment. Among all these strategies, microbial degradation is considered the most efficient and economical method (Khanal et al., 2006). 5.

(36) The abilities of steroid metabolism are diverse among microbes. Steroids transformation can be mediated by numerous microorganisms, including bacteria, yeast, fungi, and microalgae; however, the complete degradation (i.e., mineralization) of natural steroids to CO2 has been exclusively documented in bacteria (Begstrand et al., 2016; Ismail and Chiang, 2011). Numerous SHs-degrading bacteria have been isolated from a variety of environments such as human subgingival plaque, stony coral Montipora aequituberculata, ditches, soils, river sediments, and even from the deep-sea sediment (ca. 4500 m in depth) (Table 1). Based on the molecular investigations and physiological tests, SHs-degrading bacteria belong. to. the. phyla. or. classes. Actinobacteria,. Firmicutes,. Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Bacteroidetes. Bacteria depend on either oxygen or nitrate as an electron acceptor to degrade SHs and several SHs degradation pathways have been proposed.. 1.3.1 Microbial androgen degradation The aerobic degradation pathway of androgens has been elucidated in the betaproteobacterium Comamonas testosteroni [see a review by Horinouchi et al., 2012]]. Horinouchi et al. (2001, 2003, 2004a and 2004b) conducted a series of gene disruption experiments to identify androgen catabolic genes. Moreover, various catabolic intermediates were identified from the C. testosteroni mutants lacking androgen degradation genes. As 6.

(37) shown in Figure 2A, the aerobic degradation of testosterone by C. testosteroni is initiated by the dehydrogenation of the 17β-hydroxyl group to produce androst-4-en-3,17-dione (AD), which is then transformed to androsta-1,4-diene-3,17-dione (ADD). The degradation of the sterane structure begins with the introduction of a hydroxyl group at C-9 of ADD (Horinouchi et al., 2012). The resulting intermediate is extremely unstable and undergoes simultaneous cleavage of the B-ring accompanied by aromatization of the A-ring to produce a secosteroid, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA). The further cleavage of the core ring system proceeds through hydroxylation at C-4 (Horinouchi et al., 2004a), and the A-ring is then split via an extradiol dioxygenase (TesB)mediated meta-cleavage. The tesB-disrupted mutant could not grow on androgens, indicating that the dioxygenase TesB is essential for aerobic androgen degradation (Horinouchi et al., 2001). The tesB gene is embedded in a gene cluster of C. testosteroni comprising 18 androgen catabolic genes (Horinouchi et al., 2004b). The gene cluster is widely present in androgen-degrading proteobacteria, including species within the genera. Burkholderia,. Hydrocarboniphaga,. Comamonas,. Cupriavidus,. Marinobacterium,. Glaciecola,. Novosphingobium,. Pseudoalteromonas, Pseudomonas, Shewanella, and Sphingomonas (Bergstrand et al., 2016, Horinouchi et al., 2012). In addition to the wellstudied 9,10-seco pathway, the anaerobic catabolic pathway of androgens has been established in Steroidobacter (Sdo.) denitrificans DSMZ 18526 7.

(38) (Chiang et al., 2010; Fahrbach et al., 2010; Leu et al., 2011; Wang et al., 2013b). Hanselman et al. (2003) indicated that anoxic sediments and soil might be reservoirs for steroids because microbial degradation occurs slowly in these matrices. Up to now, only two anaerobic species capable of growing on androgens under nitrifying condition have been reported (Fahrbach et al., 2008, Tarlera and Denner 2003) whereas the biochemical and molecular details of androgens anaerobic degradation is limited. The first ring-cleaved intermediate of the initial androgen anaerobic degradation has been identified by Wang et al. (2013b) using Sdo. dentitrificans, a facultative anaerobic gammaproteobacterium isolated from anoxic digested sludge and can mineralize androgen under both oxic and anoxic conditions. In contrast to the aerobic pathway in which the sterane cleavage first happens at the B-ring, in anaerobic androgen catabolism of Sdo. denitrificans, the sterane cleavage first occurs in the Aring and metabolites with an aromatic A-ring are not produced. As shown in Figure 2B, in this anaerobic pathway (2,3-seco pathway) of androgen degradation, testosterone is first converted to 1-dehydrotestosterone (dT) and then reduced to produce 1-testosterone, followed by water addition to the C1/C2 double bond of 1-testosterone. Subsequently, the C-1 hydroxyl group is oxidized to produce 17-hydroxy-androstan-1,3-dione, which Aring is furtherly cleaved to produce 17-hydroxy-androstan-1-oxo-2,3-secoandrostan-3-oic acid (2,3-SAOA) (Lin et al., 2015, Wang et al., 2013a). 8.

(39) Regardless the catabolic genes and enzymes involved in this anaerobic catabolic pathway are scant, interestingly, Wang et al. (2013b) indicated that the A-ring structure transformation process is identical to the 2cyclohexenone. anaerobic. degradation. pathway. demonstrated. in. Alicycliphilus (Ali.) denitrificans (Jin et al., 2011; Figure 3). Moreover, the 2-cyclohexenone hydratase catalyzing the addition of water to the C = C bond of α, β-unsaturated carbonyl compounds was purified and characterized.. This. heterotrimeric. enzyme. (MyhADH). contains. molybdopterin, Flavin adenine dinucleotide (FAD), and [2Fe-2S] clusters and belongs to the xanthine oxidase family. Therefore, based on the structure similarity between the sequential intermediate metabolites of these two anaerobic degradation pathways, Sdo. dentitrificans might utilize homologous enzyme of MyhADH in the 2,3-seco pathway.. 1.3.2 Microbial estrogen degradation Compared with the extensive information available on aerobic catabolism of androgens (Horinouchi et al., 2012), considerably less is known regarding the biochemical mechanisms involved in aerobic estrogen biodegradation in spite of that estrogens are the most worrisome environmental endocrine-disrupting compounds among the steroid hormones. The proposed aerobic pathways of estrogen biodegradation are sporadic (Figure 4). More than 40 years ago, Coombe et al. (1966) established the first estrogen degradation pathway in an actinobacterium 9.

(40) Nocardia sp. strain E110. isolated from the soil. As the first step in the proposed pathway, E1 is transformed to 4-hydroxyestrone (4-OH-E1). The proposed 4-OH-E1 and the ring-cleaved product were not detected in that study; nevertheless, this catabolic metabolite was suggested to re-cyclize with ammonium to produce the pyridine derivative. Kurisu et al. (2010) proposed. two. other. estrogen. degradation. pathways. by. using. Sphingomonas sp. strain ED8 as the model organism. In that study, 4-OHE1 and 4-hydroxyestradiol were identified when the meta-cleavage inhibitor, 3-chlorocatechol, was present in the reaction mixtures, suggesting strain ED8 degrades estrogens through the meta-cleavage of the A-ring. The alternative pathway established in strain ED8 was initiated from a ring other than the A-ring. Nakai et al. (2011) discovered that Nitrosomonas europaea (NCIMB 11850) utilizes E2 as an only carbon source, and they identified a catabolic intermediate 16-estratetraen-3-ol (E0). It is worth mentioning that E0 was degraded by Nitrosomonas europaea, and its degradation rate was faster than that of E2. Lee and Liu (2002) incubated the supernatant of activated sludge collected from a sewage treatment plant with E2. Their batch experiments showed that E2 was degraded by sewage bacteria, leading to the production of E1. At the very early stages of E2 degradation, they detected a new metabolite (X1) containing a lactone at its D-ring. Among all the metabolites described above, only a few compounds were identified based on strong chemical evidence [e.g., using authentic 10.

(41) compounds as references or detailed mass and nuclear magnetic resonance (NMR) analyses]. Moreover, the catabolic genes and enzymes were not identified in previous studies, making an assessment of the fate and biodegradation potential of environmental estrogens challenging and unrealistic. Sphingomonas sp. strain KC8 (referred as strain KC8 hereafter), isolated from activated sludge, is a strictly aerobic alphaproteobacterium and is of particular interest for its ability to degrade E2 and E1 (Yu et al., 2007). Strain KC8 is an appropriate model organism for exploring the genes and enzymes involved in estrogen aerobic degradation. The reasons are as follow: (i) strain KC8 was able to degrade both E2 and E1 at submg/L concentrations; (ii) the 16S rRNA gene of strain KC8 was detected in the activated sludge of municipal wastewater treatment plants (2.8 x 104 to 2.8 x 105 copies ml-1) (Roh and Chu, 2010); (iii) the draft genome of strain KC8 is available (Hu et al., 2011). These three points suggest that strain KC8 is ubiquitous in municipal wastewater treatment plants and might play an important role in estrogen degradation. Anoxic river sediments and soil are considered major reservoirs for estrogens, and microbial activity is crucial for the removal of estrogens from these contaminated ecosystems (Hanselman et al., 2003). Two estrogen-degrading denitrifiers, Denitratisoma oestradiolicum and Sdo. denitrificans, were isolated from activated sludge and anoxic digested sludge, respectively (Fahrbach et al., 2006 and 2008). These denitrifiers 11.

(42) can completely degrade natural estrogens, including E1 and E2. However, none of the related catabolic intermediates have been reported thus far.. 1.3.3 Microbial progestogen degradation Literature on the biodegradation of progestogens is very limited. Liu et al. (2013) proposed the initial catabolic steps of the aerobic progesterone degradation pathway. Their metabolite analysis suggested that androgens (e.g., AD and ADD) are the catabolic intermediates of the aerobic pathway. To my knowledge, microorganisms capable of degrading progestogens under anoxic conditions have not been isolated thus far.. 1.4 Research objectives Identification of gene function in an organism is always a critical issue in life science. Even in very well-studied model organisms such as Escherichia coli there are still many genes with unknown functions (ca. 38%; Madigan et al., 2017). In general, gene knockout and recovery experiments are required to elucidate the metabolic functions or physiological roles of genes in prokaryotic cells. However, not all prokaryotes can be isolated and cultured; even if bacteria could be isolated, a successful mutagenesis is still a tremendous challenge. As a result, the information of genes and encoding enzymes involved in microbial metabolic pathways, such as those relevant to the biodegradation of SHs is limited. 12.

(43) In recent years, with the rapid development of nucleotide sequencing technology and its substantial drop in the cost, the genomic and transcriptomic analyses of prokaryotes turns into feasible. Accordingly, candidate genes associated to the biodegradation of complex substances could be identified using those cutting-edge sequencing approaches. For microorganisms in which mutagenesis is currently difficult, functions of the candidate genes can be elucidated through the isolation and characterization of the corresponding enzymes. Therefore, in the first objective of this dissertation, I used a tiered functional genomics approach to identify the catabolic genes involved in anaerobic androgen degradation by Sdo. denitrificans DSM 18526 and aerobic estrogen degradation by strain KC8. Furthermore, the functions of some essential catabolic genes were confirmed through enzyme purification and characterization, making them potential molecular markers for environmental investigations. Although SHs degradation pathways have been demonstrated in a few of bacterial isolates, almost nothing is known about the biochemical mechanisms and phylogenetic relevance of microbial SHs degradation in the environment. Culture-independent methods have been applied to study the phylogenetic identities and functions of the targeted microorganisms in complex microbial communities. Stable isotope probing (SIP) can identify microorganisms actively metabolizing the target substrates. Those active bacteria can catabolize the specific substrates labeled with stable isotopes, 13.

(44) such as. 13. C, and then incorporate and transfer those. 13. C into newly. synthesized DNA. After density-gradient centrifugation, 13C-DNA can be separated from 12C-DNA. Then, following the isolation and sequencing of isotopically labeled DNA, the microorganisms carrying out the metabolism can be identified (Dumont and Murrell 2005, Radajewski et al., 2000). Microautoradiography (MAR) and high-resolution nanometer-scale secondary-ion mass spectrometry (NanoSIMS) are also isotope-based techniques that offer the visualization of microorganisms that have incorporated a labeled substrate at a single-cell level. MAR or NanoSIMS, in conjunction with fluorescence in situ hybridization (FISH) technique become a powerful method (MAR-FISH or NanoSIMS-FISH) in the field of microbial ecology (Behrens et al., 2008, Lee et al., 1999, Musat et al., 2012, Ouverney and Fuhrman 1999). With a high lateral resolution, both techniques are used as quantitative methods to determine ecophysiology of fluorescent probe-defined single cell within complex communities. Although applications mentioned above are very powerful and have a lot of applicability, unfortunately, to study microbial degradation of SHs in the environment is still challenging and with limitations (Table 2), such as (i) completely. 13. C-labeled steroids are not commercially available; (ii) the. concentration of SHs in the environment is extremely low; (iii) the complex structure and extremely low water solubility of SHs, and (iv) inherent limitations of the available techniques. The details are as follows:. 14.

(45) Dissolved organic carbon (DOC) include steroid is the primary carbon and energy sources for heterotrophic bacteria in the aquatic environment. DOC concentrations of wastewater treatment plants are mg l-1 level, for example, the DOC in the 22 of investigated influences of wastewater treatment plants in Korea were 17-66 mg l-1 (Yang et al., 2014), 1,000 times higher than SHs. It is known that all the reported SHs-decomposing bacteria can growth by utilization of other simple organic carbon sources (Table 1). Also, completely. 13. C-labeled steroids are not commercially. available. Therefore, to stimulate the proliferation of SHs-decomposing bacteria by spiking SHs at environmental concentration level is not a feasible strategy, such as SIP. Hydrophobic compounds, such as steroids, easily attach to or pass through bacterial membranes, therefore, distinguishing the metabolic activities (passive diffusion or active uptake; redox transformation or complete degradation) of isotope-labeled microbial cells is difficult. In addition, reports indicated that some bacteria can only convert SHs but unable to utilize them, for example, Sphingomonas sp. strain KC9 and Nocardioides simplex KC3 can only convert E2 to E1 (Yu et al., 2007). So, even if bacteria with the ability to uptake steroid can be known, it is still unable to confirm which one can utilize steroids for growth in situ. Accordingly, FISH-MAR and FISH-NonaoSIMS are not appropriate methods to determine which bacteria are predominantly SHs degraders in the environment. In sum, if the catabolic metabolites of the spiked-SHs 15.

(46) within a bacterium are uncertain, identification of which bacteria are primary SHs decomposers and through which biochemical mechanisms in the environment are still with restriction. The lack of molecular markers and suitable culture-independent techniques has impeded in situ investigations of microbial steroid degradation, thus necessitating efficient and specific tools for monitoring the environmental fate of steroids. In the second objective of this dissertation, using the characteristic catabolic genes and metabolites as biomarkers, I attempted to study the biochemical mechanisms and corresponding microorganisms of steroid biodegradation in the wastewater treatment plants as well as urban rivers. To enable the substrate-derived changes in bacterial community structures, I spiked the environmental samples with high concentrations of steroid substrates (1 mM). I used the following approaches to investigate the metabolic and phylogenetic diversity of steroid biodegradation in the steroid-spiked environmental samples: (i) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identification of signature metabolites; (ii) identification of major catabolic players through next-generation sequencing techniques; and (iii) PCR-based identification of the essential catabolic genes.. 16.

(47) 2 Materials and Methods 2.1 Chemicals and bacterial strains 2.1.1 Chemicals All chemicals were analytical grade and purchased from Mallinckrodt Baker, Merck, and Sigma-Aldrich. [3,4C-13C]estrone (99%) was purchased from Cambridge Isotope Laboratories.. 18. O2 (97 atom %). was obtained from Aldrich.. 2.1.2 Bacterial strains and plasmids The bacterial strains and plasmids used in this study are summarized in Table 3.. 2.2 Bacterial cultures 2.2.1 Growth of Steroidobacter denitrificans DSM 18526 Denitrfying growth of Steroidobacter denitrificans with testosterone Sdo. denitrificans was grown with nitrate and testosterone (2.5 mM) at 28 °C in anoxic fed-batch cultures (500 ml) in the minial medium according to published procedures (Chiang et al., 2010). The basal medium was autoclaved and then complemented with separately sterilized stock solutions (Table 4).. 17.

(48) Aerobic growth of Steroidobacter denitrificans with testosterone The Sdo. denitrificans was grown in phosphate-buffered shake-flask cultures (500 ml in 2-l Erlenmeyer flasks) containing 2.5 mM testosterone or 10 mM heptanoic acid in the oxic minial medium. The basal medium was autoclaved and then complemented with the separately sterilized stock solution (Table 5). The cultures were incubated at 28˚C in an orbital shaker (180 rpm).. 2.2.2 Growth of Sphingomonas sp. strain KC8 Aerobic incubation of strain KC8 with 13C-labeled estrone Strain KC8 was first aerobically grown in R2A medium (Table 6; the final pH was adjusted to 7.0) containing 0.2 mM of unlabeled E1 (500 ml in a 2-l Erlenmeyer flask). Cells were harvested through centrifugation (7,000 ! g, 5 min, 25 °C) in the exponential growth phase at an optical density at 600 nm (OD600) of 0.5 (optical path, 1 cm). The cell pellet was re-suspended in a chemically defined medium (Table 5 but without testosterone or heptanoic acid). The cell suspension (OD600 = 5; 25 ml) was fed with 1 mM of E1 (unlabeled E1 and [3,4C-13C]E1 mixed in a 2:1 molar ratio) and was aerobically incubated at 28 °C with shaking (180 rpm). The cell suspension was sampled (1 ml) every 30 min, and testosterone (final concentration = 50 µM) was added to the culture samples as an internal control. The samples were extracted three times by using equal volumes of ethyl acetate, and the estrogen metabolites were identified through ultra18.

(49) performance. liquid. chromatography-electrospray. ionization-high. resolution mass spectrometry (UPLC–ESI–HRMS). E1 and its derivatives in the culture samples were quantified through high performance liquid chromatography (HPLC). In addition, the yeast estrogen activities of the culture samples were determined using a lacZ-based yeast estrogen assay.. Aerobic growth of strain KC8 with unlabeled steroids For the RNA sequencing (RNA-Seq) analysis, strain KC8 was first grown in R2A medium (Table 6). The KC8 cells (OD600 = 0.5) were harvested using centrifugation. The cell pellets were re-suspended in the chemically defined medium as described above. Two cell suspensions (OD600 = 1; 250 ml in 1-l Erlenmeyer flasks) were supplemented with E2 (1 mM) or testosterone (1 mM) and were incubated at 28˚C with shaking (180 rpm). The steroid substrate remaining in the bacterial cultures was quantified using HPLC. The KC8 cells were harvested using centrifugation when 0.7–0.8 mM of the substrate was consumed. For the purification of native OecC, strain KC8 was aerobically grown in R2A medium (Table 6) containing 0.5 mM of E1. The large-scale cultivation was performed in a 200-l fermentor. To produce enough amounts (approximately 1 mg) of the estrogen metabolites for the NMR analysis, strain KC8 was aerobically grown in the chemically defined medium (Table 3 but without testosterone or heptanoic acid; 500 ml in 2-l Erlenmeyer flasks) containing 2 mM of E1. The cultures 19.

(50) were incubated at 28 °C in an orbital shaker (180 rpm). The production of the estrogen metabolites in the KC8 cultures was monitored through thin layer chromatography (TLC). The cultures were subsequently extracted with ethyl acetate to recover the estrogen metabolites from the aqueous phase. Ethyl acetate extracts were separated through TLC and HPLC sequentially. The structures of HPLC-purified intermediates were elucidated through NMR spectroscopy and mass spectrometry.. 2.2.3 Other bacteria Ali. denitrificans was grown with cyclohexanol under denitrifying condition as described in Jin et al. (2011). Thauera terpenica was anaerobically grown on acetate (16 mM) and testosterone (1 mM) in the minimal medium DSMZ Medium 1315. Geobacillus kaustophilus and Hydrogenophaga pseudoflava were aerobically grown on nutrient agar (DSMZ Medium 1; Table 7) at 55 °C and on trypticase soy broth agar (DSMZ Medium 535; Table 8) at 30 °C, respectively. The other aerobic bacteria in Table 3 were aerobically cultured in Luria-Bertani (LB) medium (BD Biosciences) at 30˚C.. 2.3 Preparation of cell extracts Unless otherwise stated, crude cell extracts were prepared as follows. 20 g of frozen bacterial cells was suspended in 40 ml of 20 mM Tris-HCl buffer (pH 7.8). A French pressure cell (Thermo Fisher Scientific) was used 20.

(51) to break the bacterial cells. The cell lysate was fractionated by two steps of centrifugation: the first step involved centrifugation for 30 min at 20,000 ! g to remove cell debris. The supernatant was then centrifuged at 100,000 ! g for 2 h to separate soluble proteins from membrane-bound proteins.. 2.4 Determination of activity of sex steroid hormones 2.4.1 lacZ-based yeast androgen bioassay The sewage samples (0.5 ml) were extracted three times using equal volume of ethyl acetate. After the solvent evaporated, the extracts were redissolved in the same volume of dimethyl sulfoxide (DMSO), and the androgenic activity in the sewage samples was determined using a lacZbased yeast androgen assay. The yeast androgen bioassay was conducted as described by Fox et al. (2008) with slight modifications. Briefly, the individual steroid standards (testosterone, 1-testosterone, AD, ADD, 3HAS and E1) or sewage extracts were dissolved in DMSO, and the final concentration of DMSO in the assays (200 µl) was 1 % (v/v). The resulting DMSO solutions (2 µl) were added to yeast cultures (198 µl, initial OD600nm = 0.5) located in a 96-well microtiter plate. The β-galactosidase activity was determined after 18-h incubation at 30 ˚C. The yeast suspension (25 µl) was added to a Z buffer (225 µl) containing o-nitrophenol-β-Dgalactopyranoside (2 mM), and the reaction mixtures were incubated at 37 ˚C for 30 min. The reactions were stopped by adding 100 µl of 1 M sodium carbonate, and the amount of yellow-colored nitrophenol product was 21.