Identification of the EdcR estrogen-dependent repressor in Caenibius tardaugens NBRC 16725: construction of a cellular estradiol biosensor

genes G C A T T A C G G C A T Article Identification of the EdcR Estrogen-Dependent Repressor in Caenibius tardaugens NBRC 16725: Construction of a Cellular Estradiol Biosensor Juan Ibero, Beatriz Galán and JoséL. García *   Citation: Ibero, J.; Galán, B.; García, J.L. Identification of the EdcR Estrogen-Dependent Repressor in Caenibius tardaugens NBRC 16725: Construction of a Cellular Estradiol Biosensor. Genes 2021,12, 1846. https://doi.org/10.3390/ genes12121846 Academic Editors: Manuel Carmona and Gonzalo Durante-Rodríguez Received: 25 October 2021 Accepted: 21 November 2021 Published: 23 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Center for Biological Research Margarita Salas, Department of Microbial and Plant Biotechnology, Spanish National Research Council, Ramiro de Maeztu 9, 28040 Madrid, Spain; [email protected] (J.I.); [email protected] (B.G.) *Correspondence: jlgar[email protected] Abstract: In this work, Caenibius tardaugens NBRC 16725 (strain ARI-1) (formerly Novosphingobium tardaugens) was isolated due to its capacity to mineralize estrogenic endocrine disruptors. Its genome encodes the edc genes cluster responsible for the degradation of 17 β -estradiol, consisting of two putative operons (OpA and OpB) encoding the enzymes of the upper degradation pathway. Inside the edc cluster, we identified the edcR gene encoding a TetR-like protein. Genetic studies carried out with C. tardaugens mutants demonstrated that EdcR represses the promoters that control the expression of the two operons. These genetic analyses have also shown that 17 β -estradiol and estrone, the second intermediate of the degradation pathway, are the true effectors of EdcR. This regulatory system has been heterologously expressed in Escherichia coli, foreseeing its use to detect estrogens in environmental samples. Genome comparisons have identified a similar regulatory system in the edc cluster of Altererythrobacter estronivorus MHB5, suggesting that this regulatory arrangement has been horizontally transferred to other bacteria. Keywords: Caenibius tardaugens; estrogen; 17 β -estradiol catabolism; estrogen metabolism; transcriptional regulation; TetR regulator; estrogen biosensor; Escherichia coli 1. Introduction Endocrine disruptors (EDCs) are chemical contaminants that interfere with the endocrine system and produce adverse effects in both humans and wildlife. The exposure to estrogens, in concentrations as low as 1 ng/L, has been reported to cause feminization, decreased expression of secondary sex characteristics and a reduction in egg fertilization in fish and amphibians [ 1 ]. Natural estrogens and androgens enter the environment through the excretions of humans, domestic or farm animals and wildlife. The 17 β -estradiol (E2) is a ubiquitous pollutant usually found in soil and water systems [ 2 – 4 ]. The complete mineralization of estrogens to CO2can be accomplished aerobically [5–14] or anaerobically [15] by a limited number of bacteria, mainly from the phyla Proteobacteria and Actinobacteria. Different biochemical and genetic studies have assigned function to many catabolic genes involved in estrogen catabolism, but numerous issues concerning the degradative pathway and its regulation remain to be fully elucidated [10,16–19]. Caenibius tardaugens (formerly Novosphingobium tardaugens) is an aerobic bacterium, isolated from a sewage treatment plant in Tokyo due to its capacity to mineralize E2 and other EDCs [ 5 , 20 ]. Taking advantage of the fact that the genome of this bacterium has been recently assembled in a single contig [21], we have used it as a model system to study the degradation pathways that mineralize EDCs [22,23] (Figure 1). Genes 2021,12, 1846. https://doi.org/10.3390/genes12121846 https://www.mdpi.com/journal/genes Genes 2021,12, 1846 2 of 20 Genes 2021, 12, x FOR PEER REVIEW 2 of 21 Figure 1. Proposed estrogen degradation pathway in C. tardaugens NBRC 16725. Compound names are indicated with an abbreviation: (E2) estradiol; (E1) estrone; (4-OHE1) 4-hydroxystrone; (HIP) 3aα-H -4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione. Enzyme names are: (17β-hsd) 3β, 17β-hydroxysteroid dehydrogenase; (edcA) E1 4-hydroxylase; (edcB) 4-OHE1 4,5-dioxygenase; (edcC) meta-cleavage product decarboxylase. The catalytic genes of C. tardaugens are indicated in italics, with the nomenclature EGO55_xxxxx. These studies have allowed us to identify two gene clusters, named SD and edc, responsible for the degradation of testosterone (TES) and E2, respectively [22,23]. The transcriptomic analysis enabled the characterization of the edc cluster that is organized in two divergent operons, OpA (EGO55_13525–EGO55_13565) and OpB (EGO55_13570– EGO55_13600), and a divergently expressed gene, EGO55_13520, which encodes a putative TetR-like transcriptional regulator [23] (Figure 2). The E2 degradation pathway is tightly regulated, reinforcing the idea that the TetR-like protein could play a regulatory role [23]. The TetR family of regulators is a large family of one-component bacterial signal transduction systems and their regulatory mechanisms, as well as their large variety of effectors, have been extensively studied [24,25]. Although some of these regulators have been proposed to be involved in the metabolism of steroid compounds in bacteria, none have been experimentally shown to interact specifically with estrogens (see discussion). In this work, we demonstrate that EdcR regulates the edc cluster in C. tardaugens. This regulator acts as a repressor of the expression of the edc cluster genes. We determined that E2 and E1 work as effectors to de-repress the system. By heterologously expressing this regulatory system in Escherichia coli, we constructed a cell biosensor to detect the presence of E2 and E1 in environmental samples. Figure 1. Proposed estrogen degradation pathway in C. tardaugens NBRC 16725. Compound names are indicated with an abbreviation: (E2) estradiol; (E1) estrone; (4-OHE1) 4-hydroxystrone; (HIP) 3a α -H -4 α (3 0 -propanoate)-7a β -methylhexahydro-1,5-indanedione. Enzyme names are: (17 β -hsd) 3 β , 17 β -hydroxysteroid dehydrogenase; (edcA) E1 4-hydroxylase; (edcB) 4-OHE1 4,5-dioxygenase; (edcC) meta-cleavage product decarboxylase. The catalytic genes of C. tardaugens are indicated in italics, with the nomenclature EGO55_xxxxx. These studies have allowed us to identify two gene clusters, named SD and edc, responsible for the degradation of testosterone (TES) and E2, respectively [ 22 , 23 ]. The transcriptomic analysis enabled the characterization of the edc cluster that is organized in two divergent operons, OpA (EGO55_13525–EGO55_13565) and OpB (EGO55_13570– EGO55_13600), and a divergently expressed gene, EGO55_13520, which encodes a putative TetR-like transcriptional regulator [ 23 ] (Figure 2). The E2 degradation pathway is tightly regulated, reinforcing the idea that the TetR-like protein could play a regulatory role [ 23 ]. The TetR family of regulators is a large family of one-component bacterial signal transduction systems and their regulatory mechanisms, as well as their large variety of effectors, have been extensively studied [ 24 , 25 ]. Although some of these regulators have been proposed to be involved in the metabolism of steroid compounds in bacteria, none have been experimentally shown to interact specifically with estrogens (see discussion). Genes 2021, 12, x FOR PEER REVIEW 2 of 21 Figure 1. Proposed estrogen degradation pathway in C. tardaugens NBRC 16725. Compound names are indicated with an abbreviation: (E2) estradiol; (E1) estrone; (4-OHE1) 4-hydroxystrone; (HIP) 3aα-H -4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione. Enzyme names are: (17β-hsd) 3β, 17β-hydroxysteroid dehydrogenase; (edcA) E1 4-hydroxylase; (edcB) 4-OHE1 4,5-dioxygenase; (edcC) meta-cleavage product decarboxylase. The catalytic genes of C. tardaugens are indicated in italics, with the nomenclature EGO55_xxxxx. These studies have allowed us to identify two gene clusters, named SD and edc, responsible for the degradation of testosterone (TES) and E2, respectively [22,23]. The transcriptomic analysis enabled the characterization of the edc cluster that is organized in two divergent operons, OpA (EGO55_13525–EGO55_13565) and OpB (EGO55_13570– EGO55_13600), and a divergently expressed gene, EGO55_13520, which encodes a putative TetR-like transcriptional regulator [23] (Figure 2). The E2 degradation pathway is tightly regulated, reinforcing the idea that the TetR-like protein could play a regulatory role [23]. The TetR family of regulators is a large family of one-component bacterial signal transduction systems and their regulatory mechanisms, as well as their large variety of effectors, have been extensively studied [24,25]. Although some of these regulators have been proposed to be involved in the metabolism of steroid compounds in bacteria, none have been experimentally shown to interact specifically with estrogens (see discussion). In this work, we demonstrate that EdcR regulates the edc cluster in C. tardaugens. This regulator acts as a repressor of the expression of the edc cluster genes. We determined that E2 and E1 work as effectors to de-repress the system. By heterologously expressing this regulatory system in Escherichia coli, we constructed a cell biosensor to detect the presence of E2 and E1 in environmental samples. Figure 2. Scheme of the C. tardaugens NBRC 16725 estrogen degradation cluster (edc). Operons OpA, OpB and the intergenic region are depicted with a line: red, purple and yellow, respectively. Genes annotated as tetR (EGO55_13520), cytochrome P450 hydroxylase (EGO55_13525), hydratase (EGO55_13530), lipid-transfer protein (EGO55_13535), enoyl-CoA hydratase/ isomerase (EGO55_13540), 2-ketoacyclohexanecarboxyl-CoA (EGO55_13545), acetyl-CoA acyltransferase (EGO55_13550), hydroxymethylglutaryl-CoA synthase (EGO55_13555), 3-hydroxyacyl-CoA dehydrogenase (EGO55_13560), acyl-CoA dehydrogenase (EGO55_13565), 4-hydroxyestrone-4,5-dioxygenase (EGO55_13570), vicinal oxygen chelate containing protein (EGO55_13575), indolepyruvate ferredoxin oxidoreductase (EGO55_13580), acyl-CoA dehydrogenase (EGO55_13585), MaoC dehydratase (EGO55_13590), MaoC dehydratase (EGO55_13595) and TonB dependent receptor (EGO55_13600) are also shown. Genes 2021,12, 1846 3 of 20 In this work, we demonstrate that EdcR regulates the edc cluster in C. tardaugens. This regulator acts as a repressor of the expression of the edc cluster genes. We determined that E2 and E1 work as effectors to de-repress the system. By heterologously expressing this regulatory system in Escherichia coli, we constructed a cell biosensor to detect the presence of E2 and E1 in environmental samples. 2. Materials and Methods 2.1. Chemicals Testosterone (TES), 17 β -estradiol (E2), estrone (E1), 4-hydroxyestrone (4-OHE1), estriol (E3), ethinylestradiol (EE2), pyruvate (Pyr), chloroform, n-hexane, ethyl acetate, sulphuric acid and acetonitrile were purchased from Sigma (Steinheim, Germany). Randomly methylated β -cyclodextrin (TRMB-T Randomly Methylated BCD) (CDX) was purchased from Cyclodex (Alachua, FL, United States). 2.2. Strains and Growth Media Bacterial strains and plasmids used in this study are listed in Table 1.C. tardaugens NBRC 16725 (strain ARI-1) was purchased from the Leibniz-Institut DSMZ type culture collection. Nutrient broth (NB) (Difco TM , Burlington, NJ, USA) was used as rich medium to grow this strain at 30 ◦ C in an orbital shaker at 200 rpm. This strain was also cultured in minimal medium M63 [KH 2 PO 4 (136 g/L), (NH 4 ) 2 SO 4 (20 g/L), FeSO 4· 7H 2 O (5 mg/L), pH 7.0] supplemented with 0.39 mM CaCl 2 , 1 mM MgSO 4 and different carbon sources. Steroids and pyruvate stock solutions were prepared in PBS buffer and 70 mM CDX to reach a final carbon concentration of 54 mM in 20 mM CDX in the culture medium. Escherichia coli DH10B was grown at 37 ◦ C in an orbital shaker at 200 rpm in lysogeny broth (LB) medium [ 26 ]. The appropriate antibiotics, chloramphenicol (34 µ g/mL), kanamycin (50 µg/mL) or rifampicin (50 µg/mL) were added when needed. Table 1. Bacterial strains and plasmids used in this study. Strains Genotype and Characteristics Source/Reference C. tardaugens NBRC 16725 wild type strain (ARI-1) [20] RfRRfRstrain efficient for conjugation [22] ∆edcA C. tardaugens NBRC 16725 ∆EGO55_13525 [23] ∆edcR C. tardaugens NBRC 16725 ∆EGO55_13520 This study E. coli DH10B F−,mcrA, ∆(mrr hsdRMS-mcrBC), Φ80dlacZ∆M15, ∆lacX74,deoR,recA1,araD139, ∆(ara-leu)7697, galU,galK,λ−,rpsL,endA1,nupG Invitrogen HB101 supE44, ara14, galK2, leuB, lacY1, ∆ (gpt-proA)62, rpsL20, xyl-5, mtl-1, recA13, ∆ (mcrC-mrr), hsdS20 (rB−mB−), SmR[27] Plasmids pK18mobsacB KmR, ColE oriV, Mob+, lacZα,sacB; vector for allelic exchange homologous recombination mutagenesis [28] pK18edcR pK18mobsacB derivative used for EGO55_13520 deletion This study pSEVA237PlexA KmR, oriV (pBBR1), constituve expression of gfp gene under the control of the PlexA promoter [29] pSEVA23PlexA pSEVA237PlexA where gfp was deleted using XbaI-SpeI restriction enzymes and served as empty vector [23] pSEVA23edcR pSEVA237PlexA where gfp gene was replaced by EGO55_13520 This study pSEVA237Pa pSEVA237PlexA where PlexA promoter was replaced by PaThis study pSEVA237Pb pSEVA237PlexA where PlexA promoter was replaced by PbThis study pSEVA237Pt pSEVA237PlexA where PlexA promoter was replaced by PtThis study pSEVA237M-BCD2-14g KmR, pSEVA237M derivative, synthetic bicistronic RBS BCD2, P14g promoter Kindly provided by P. Nikel pSEVA237MPb KmR, pSEVA237M-BCD2-14g derivative where P14g promoter was replaced by PbThis study pSEVA651 GmR,oriV (RSF1010), standard MCS [30] pSEVA65edcR pSEVA651 containing EGO55_13520 gene expressed constitutively under P lexA promoter This study KmR, kanamycin resistance gene. Genes 2021,12, 1846 4 of 20 2.3. DNA Manipulation Molecular biology and DNA manipulations were performed as described elsewhere [ 27 ]. C. tardaugens genomic DNA was extracted as described before [ 21 ]. Plasmid DNA was purified using High Pure Plasmid Isolation Kit (Roche, Basel, Switzerland). DNA fragments were purified with QIAquick PCR Purification Kit (Qiagen, Düsseldorf, Germany) or QIAquick Gel Extraction Kit (Qiagen, Düsseldorf, Germany). E. coli cells were transformed using the RbCl method or by electroporation (Gene Pulser; Bio-Rad, Hercules, USA) [ 31 ]. DNA amplification was performed in a Mastercycler Gradient (Eppendorf, Hamburg, Germany) using the oligonucleotides listed in Table S1, which were purchased from Sigma (Steinheim, Germany). Phusion High-Fidelity DNA Polymerase (New England Biolabs (NEB), Ipswich, MA, USA) was used for cloning amplifications and Taq DNA polymerase (Biotools, Madrid, Spain) for screening and RT-PCR assays. All PCR products were checked by agarose gel electrophoresis and those aimed for cloning were confirmed by DNA sequencing by Secugen S.L. (Madrid, Spain). Digestion of DNA fragments was done using restriction enzymes from NEB and ligation was performed with Instant Sticky-end Ligase Master Mix (NEB). Construction of pSEVA237MPb was performed as follows: first digesting pSEVA237M-BCD2-14g with PacI and AvrII restriction enzymes to excise 14 g promoter; then digesting pSEVA237Pb-edcA [ 23 ] using PacI and XbaI restriction enzymes to extract P b promoter and finally ligating the core of pSEVA237M-BCD2 plasmid with P b promoter fragment (Figure S1). pSEVA65edcR plasmid was constructed digesting pSEVA23edcR with PacI and SpeI restriction enzymes to extract a DNA fragment containing P lexA promoter and edcR gene and ligating it into pSEVA651 plasmid backbone. 2.4. RNA Manipulation Total RNA of C. tardaugens cells was extracted from 12 ml of cultures grown in minimal medium with 20 mM CDX and TES or pyruvate as carbon sources. Cells were harvested in mid exponential phase (OD 600 0.6) and stored at − 80 ◦ C. Pellets were thawed and cells were lysed in 400 µ L TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) containing lysozyme (50 mg/mL) following three freezing/thawing cycles. High Pure Isolation Kit (Roche, Basel, Switzerland), followed by DNA-free TM DNA Removal Kit (Invitrogen, Waltham, MA, USA) treatment, was used to obtained pure RNA. Purity and concentration were measured in a ND1000 spectrophotometer (Nanodrop Technologies, Thermo Fisher Scientific, Waltham, MA, USA). cDNA used as the template for RT-PCR was obtained with a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) following the manufacturer’s instructions, where 1 µ g of total RNA was used as the template in 20 µ L of reverse transcription reaction. RT-PCR was performed adding 1 µ L of cDNA, 0.5 mM of each oligonucleotide, 0.25 mM dNTPs, 5% DMSO (v/v) and 1 U of Taq DNA polymerase to a reaction mixture of 30 µ L in a 20-cycle PCR. Oligonucleotides used in RT-PCR expression analysis are listed in Table S1. To check that RNA was not contaminated with DNA, a 30-cycle PCR was performed using 200 ng total RNA as template and oligonucleotides 5RTRecAf and 5RTRecAr (Table S2). 2.5. Construction of C. tardaugens Knockout Strains The knockout strains were constructed by double homologous recombination using the suicide vector pK18mobsacB [ 28 ] as described before [ 22 ]. C. tardaugens genomic DNA was used as template to amplify two fragments of ≈ 700 bp containing the upstream and downstream regions of the gene to delete UP and DOWN fragments (Tables Table 1and S1), respectively. The fragments were digested with the appropriate restriction enzymes and cloned in the unique sites of the plasmid. The ligation product was transformed into E. coli DH10B competent cells and once recombinant candidates were PCR-checked, the cloned region was confirmed by sequencing. The plasmids were transformed by triparental conjugation [ 32 ] into C. tardaugens Rf R as recipient strain using E. coli HB101 (pRK600) [ 33 ] as a helper and E. coli DH10B, harboring the corresponding vector, as a donor, as described before [23]. Genes 2021,12, 1846 5 of 20 2.6. Complementation of C. tardaugens Knockout Strains The mutant strains were transformed with pSEVA23 plasmids harboring the corresponding deleted genes under the expression control of P lexA constitutive promoter [ 29 ] (Table 1) using triparental conjugation, following the same protocol used to construct C. tardaugens knockout strains. 2.7. Fluorescence Measurement of Cultures on a Plate Reader The fluorescence signal of GFP (Green Fluorescent Protein) in cultures of strains carrying transcriptional fusions was measured using a Varioskan Flash microplate reader (Thermo Scientific, Waltham, MA, USA), using excitation and emission wavelengths of 485 nm and 511 nm, respectively. The measurements were made in 96-well plates and three technical replicates of each sample were taken into three different wells. The measurement was performed by loading into each well 200 µ L of cells at an OD 600 of 1.0, previously washed with 0.85% (w/v) NaCl solution in H 2 Omq. The measurements were normalized to OD and to a control where no GFP (only 0.85% (w/v) NaCl solution in H2Omq) was used. 2.8. In Silico Genomic Analysis Analysis of nucleotide sequences, chromatograms from sequencing reactions, design of genetic engineering experiments, design and analysis of oligonucleotides and mapping of reads obtained from transcriptome sequencing against the C. tardaugens genome, were done with the program Geneious R11.1.5 (https://www.geneious.com, accessed on 25 October 2021). Identification of conserved promoter sequences with was performed using the tools BPROM [ 34 ] from the Softberry server (https://www.softberry.com, accessed on 25 October 2021) and SAPPHIRE [ 35 ] (https://www.biosapphire.com, accessed on 25 October 2021). Gene product prediction was done with Rapid Annotations using Subsystems Technology (RAST) [ 36 ]. Amino acid sequences of individual proteins were compared with those in the databases by using the BLASTp program [ 37 ] from the National Center for Biotechnology Information (NCBI; https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 25 October 2021) server. Multiple alignments of nucleotide and amino acid sequences were performed with ClustalOmega [ 38 ] from the server of the European Bioinformatics Institute (EMBL-EBI; http://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 25 October 2021). 3. Results 3.1. In Silico Analysis of the Main Promoters Responsible for the Expression of C. tardaugens Edc Gene Cluster Transcriptomic studies have revealed a high induction level of the edc cluster genes in the presence of E2 [ 23 ]. The structure of the edc cluster suggests the existence of two promoters in the intergenic region located between the divergent OpA and OpB putative operons, and at least another promoter upstream the EGO55_13520 (edcR) gene, encoding a putative regulatory protein EdcR that is divergently transcribed to the OpA genes (Figure 2). Furthermore, the large intergenic region of 85 bp between the EGO55_13595 and EGO55_13600 genes suggests the presence of another promoter region that can drive the expression of the EGO55_13600 (edcT) gene, encoding a putative transporter (Figure 2). To get a whole picture of the edc gene expression, the raw reads obtained in the RNAseq experiments carried out in the presence of E2 or Pyr were mapped against the genome sequence. As expected, the result reflected a higher number of reads when cells were grown in the presence of E2, which corresponds to the higher level of induction already described (Figure 3). In both conditions, the intergenic region between the EGO55_13565 and EGO55_13570 genes (i.e., the initial genes of the OpA and OpB divergent operons, respectively) showed a low number of aligned reads (i.e., an aligning gap) compared with the reads aligned to both catabolic genes, suggesting that this region could contain the two promoters which drive the expression of both operons (Figure 3). A less pronounced aligning gap, but still significant, was observed upstream of the EGO55_13520 gene suggesting the existence of another promoter region (Figure 3). Genes 2021,12, 1846 6 of 20 Genes 2021, 12, x FOR PEER REVIEW 7 of 21 To get a whole picture of the edc gene expression, the raw reads obtained in the RNAseq experiments carried out in the presence of E2 or Pyr were mapped against the genome sequence. As expected, the result reflected a higher number of reads when cells were grown in the presence of E2, which corresponds to the higher level of induction already described (Figure 3). In both conditions, the intergenic region between the EGO55_13565 and EGO55_13570 genes (i.e., the initial genes of the OpA and OpB divergent operons, respectively) showed a low number of aligned reads (i.e., an aligning gap) compared with the reads aligned to both catabolic genes, suggesting that this region could contain the two promoters which drive the expression of both operons (Figure 3). A less pronounced aligning gap, but still significant, was observed upstream of the EGO55_13520 gene suggesting the existence of another promoter region (Figure 3). Figure 3. Scheme of gene expression of estrogen degradation cluster (edc) in C. tardaugens NBRC 16725 cells when grown on E2 compared to PYR condition. (a) Mapping of RNA-Seq raw reads against edc cluster sequence obtained in PYR and E2 conditions. (b) Scheme of edc cluster. (c) Induction levels of the edc cluster measured as fold change (FC). Interestingly, Figure 3 also shows that in the Pyr condition, the number of reads mapped at the EGO55_13520 (edcR) gene was proportionally higher than the average of the reads mapped to the rest of the catabolic genes, suggesting that this gene was more expressed than the other genes of the edc cluster in the absence of E2. When the expression of the edcR regulatory gene was more precisely compared with the expression of the other genes of edc cluster using the number of normalized reads, we observed that the average of normalized reads mapped to EGO55_13520 gene when cells are cultured on Pyr (1553 transcripts per kilobase million, TPMs) was higher than the reads mapped to OpA (239 TPMs) and OpB (889 TPMs) operons [23]. However, when cells are cultured on E2, edcR had slightly less reads per transcription unit (9390 TPMs) than the reads of OpA (14401 TPMs) and OpB (22708 TPMs) operons [23]. This suggests that EdcR might act as a repressor during growth on Pyr, a condition in which the genes of the edc cluster are silenced. It is also worth mentioning that Figure 3 does not show an aligning gap at the end of the OpB operon and the beginning of the EGO55_13600 (edcT) gene, likely suggesting that this gene can be expressed not only by its own promoter, but also by the promoter of the OpB operon. On the other hand, the transcriptomic analysis revealed a higher induction of the genes of the OpA operon compared to those of the OpB operon, likely due to an overall low expression level in PYR condition of genes of the OpA operon (239 TPMs) compared to those of the OpB operon (889 TPMs) (Figure 3c). Furthermore, there is a significant difference in the expression levels between the EGO55_13595 gene belonging to the OpB Figure 3. Scheme of gene expression of estrogen degradation cluster (edc) in C. tardaugens NBRC 16725 cells when grown on E2 compared to PYR condition. ( a ) Mapping of RNA-Seq raw reads against edc cluster sequence obtained in PYR and E2 conditions. ( b ) Scheme of edc cluster. (c) Induction levels of the edc cluster measured as fold change (FC). Interestingly, Figure 3also shows that in the Pyr condition, the number of reads mapped at the EGO55_13520 (edcR) gene was proportionally higher than the average of the reads mapped to the rest of the catabolic genes, suggesting that this gene was more expressed than the other genes of the edc cluster in the absence of E2. When the expression of the edcR regulatory gene was more precisely compared with the expression of the other genes of edc cluster using the number of normalized reads, we observed that the average of normalized reads mapped to EGO55_13520 gene when cells are cultured on Pyr (1553 transcripts per kilobase million, TPMs) was higher than the reads mapped to OpA (239 TPMs) and OpB (889 TPMs) operons [ 23 ]. However, when cells are cultured on E2, edcR had slightly less reads per transcription unit (9390 TPMs) than the reads of OpA (14401 TPMs) and OpB (22708 TPMs) operons [ 23 ]. This suggests that EdcR might act as a repressor during growth on Pyr, a condition in which the genes of the edc cluster are silenced. It is also worth mentioning that Figure 3does not show an aligning gap at the end of the OpB operon and the beginning of the EGO55_13600 (edcT) gene, likely suggesting that this gene can be expressed not only by its own promoter, but also by the promoter of the OpB operon. On the other hand, the transcriptomic analysis revealed a higher induction of the genes of the OpA operon compared to those of the OpB operon, likely due to an overall low expression level in PYR condition of genes of the OpA operon (239 TPMs) compared to those of the OpB operon (889 TPMs) (Figure 3c). Furthermore, there is a significant difference in the expression levels between the EGO55_13595 gene belonging to the OpB operon and EGO55_13600 gene, suggesting, as pointed out above, the presence of an additional promoter element driving the expression of the EGO55_13600 gene (Figure 3a,c). Therefore, these analyses support the existence of at least four promoter regions in the edc cluster, i.e., P r ,P t , P a and P b , to control transcription of edcR and edcT genes, and OpA and OpB operons, respectively (Figure 3). The promoter regions were analyzed to detect the putative RNA polymerase binding sequences (boxes -10 and -35), as well as the possible operator binding sequences for transcriptional factors using bioinformatic tools (see Materials and Methods). Possible -10 and -35 boxes were identified in these regions, but the boxes of P b promoter were more similar to the consensus -10 (TATAAT) and -35 (TTGACA) boxes of E. coli [ 39 ] (Figure 4). On the other hand, a conserved palindromic sequence (TnnCnnTACnAnTnGTAnnGnnA) was identified in the P a and P b promoters as a putative operator site. A similar but less conserved sequence was also identified at P r and P t ‘promoters. This putative operator sequence is overlapping the -10 box in all these promoters (Figure 4). Genes 2021,12, 1846 7 of 20 Genes 2021, 12, x FOR PEER REVIEW 8 of 21 operon and EGO55_13600 gene, suggesting, as pointed out above, the presence of an additional promoter element driving the expression of the EGO55_13600 gene (Figures 3a,c). Therefore, these analyses support the existence of at least four promoter regions in the edc cluster, i.e., P r , P t , P a and P b , to control transcription of edcR and edcT genes, and OpA and OpB operons, respectively (Figure 3). The promoter regions were analyzed to detect the putative RNA polymerase binding sequences (boxes -10 and -35), as well as the possible operator binding sequences for transcriptional factors using bioinformatic tools (see Materials and Methods). Possible -10 and -35 boxes were identified in these regions, but the boxes of P b promoter were more similar to the consensus -10 (TATAAT) and -35 (TTGACA) boxes of E. coli [39] (Figure 4). On the other hand, a conserved palindromic sequence (TnnCnnTACnAnTnGTAnnGnnA) was identified in the P a and P b promoters as a putative operator site. A similar but less conserved sequence was also identified at P r and P t ` promoters. This putative operator sequence is overlapping the -10 box in all these promoters (Figure 4). When the promoter regions of C. tardaugens were compared with the equivalent regions of the edc homologous gene clusters of the estrogen-degrading bacteria Altererythrobacter estronivorus MHB5 and Sphingomonas sp. KC8 [10,14], we observed only highly conserved sequences in the C. tardaugens and A. estronivorus MHB5 clusters (Figure 4). Figure 4. Analysis of promoter sequences of the edc cluster. Sequences of the P a , P b , P t and P r regions of C. tardaugens NBRC 16725 and P a and P b of A. estronivorus MHB5. The -35 and -10 putative boxes are shown in red and purple, respectively. The start codons of the downstream gene are highlighted in pink and those of the gene upstream of the promoter in green, and the palindromic sequences identified are highlighted in gray. Identified palindromes are marked with yellow arrows and nucleotides that differ from the palindrome are marked with yellow stars. 3.2. Analysis of the EdcR Function The edcR gene (EGO55_13520) has been annotated as a transcriptional regulator of the TetR family (Figure 2) according to pfam containing the characteristic domains of the proteins of this family [24,25], i.e., a DNA-binding domain (helix turn helix HTH, PF00440) and a C-terminal domain usually involved in inducer binding and protein oligomerization (domain TetR_C_11, PF16859). The edc cluster of A. estronivorus MHB5 contains a MB02_RS00150 gene encoding a protein that shares 95% amino acid identity at the DNA binding domain (Figure 5) [14]. However, the product of the homologous KC8_05330 gene from Sphingomonas sp. KC8 shows only 53% amino acid identity at the DNA binding domain (Figure 5). This result suggests that the regulator of KC8 might recognize a different operator sequence and supports the exitance of conserved operator sequences in C. tardaugens and A. estronivorus. Figure 4. Analysis of promoter sequences of the edc cluster. Sequences of the P a ,P b ,P t and P r regions of C. tardaugens NBRC 16725 and P a and P b of A. estronivorus MHB5. The -35 and -10 putative boxes are shown in red and purple, respectively. The start codons of the downstream gene are highlighted in pink and those of the gene upstream of the promoter in green, and the palindromic sequences identified are highlighted in gray. Identified palindromes are marked with yellow arrows and nucleotides that differ from the palindrome are marked with yellow stars. When the promoter regions of C. tardaugens were compared with the equivalent regions of the edc homologous gene clusters of the estrogen-degrading bacteria Altererythrobacter estronivorus MHB5 and Sphingomonas sp. KC8 [ 10 , 14 ], we observed only highly conserved sequences in the C. tardaugens and A. estronivorus MHB5 clusters (Figure 4). 3.2. Analysis of the EdcR Function The edcR gene (EGO55_13520) has been annotated as a transcriptional regulator of the TetR family (Figure 2) according to pfam containing the characteristic domains of the proteins of this family [ 24 , 25 ], i.e., a DNA-binding domain (helix turn helix HTH, PF00440) and a C-terminal domain usually involved in inducer binding and protein oligomerization (domain TetR_C_11, PF16859). The edc cluster of A. estronivorus MHB5 contains a MB02_RS00150 gene encoding a protein that shares 95% amino acid identity at the DNA binding domain (Figure 5) [ 14 ]. However, the product of the homologous KC8_05330 gene from Sphingomonas sp. KC8 shows only 53% amino acid identity at the DNA binding domain (Figure 5). This result suggests that the regulator of KC8 might recognize a different operator sequence and supports the exitance of conserved operator sequences in C. tardaugens and A. estronivorus. Genes 2021, 12, x FOR PEER REVIEW 9 of 21 Figure 5. Sequence alignment of the regulators encoded by EGO55_13520, MB02_RS00150 and KC8_05330, in C. tardaugens NBRC 16725 (CT), A. estronivorus MHB5 (AE) and Sphingomonas sp. KC8 (KC8), respectively. DNA-binding domains (a,c) and effector-binding domains (b,d). Identical and conserved amino acids are marked with "*" and “:”, respectively. To study the role of EdcR in the metabolism of E2 in C. tardaugens, we constructed a ∆edcR deletion mutant that was cultured it in minimal medium supplemented with E2 as the sole carbon and energy source. The ∆edcR strain is able to grow in E2 faster than the wild-type strain (Figure 6a), suggesting that EdcR is not an activator of E2 degradation. When the ∆edcR mutant was complemented with plasmid pSEVA23edcR expressing the gene edcR under the control of the P lexA constitutive promoter (Table 1), the recombinant strain grew slower than the ∆edcR mutant (Figure 6b), suggesting a probable repressor activity of the EdcR protein. Figure 6. Growth curves (OD 600 ) of C. tardaugens strains growing in minimal medium supplemented with 2 mM E2. (a) C. tardaugens wt strain and C. tardaugens ΔedcR mutant. (b) C. tardaugens ΔedcR (pSEVA23PlexA) (ΔedcR-v) and C. tardaugens ΔedcR (pSEVA23edcR) (ΔedcR-c). The values correspond to the mean of three independent biological replicas (n = 3) and error bars correspond to SD. The influence of the absence of edcR on the expression of the genes of the edc cluster was determined using the ∆edcR mutant strain. Total RNA was extracted from the cells of wild-type and ∆edcR mutant strains growing in minimal medium with E2 or TES as the only carbon and energy sources, and RT-PCRs were performed to determine the relative expression of some representative genes of the edc cluster. EGO55_13555, EGO55_13570 and EGO55_13600 genes were chosen as representative markers of the expression of the OpA and OpB operons. The housekeeping recA gene (EGO55_01665) was used as internal control. As expected, the expression of the genes was detected in the presence of E2 in the wild-type and ∆edcR mutant strains (Figure 7). However, in the presence Figure 5. Sequence alignment of the regulators encoded by EGO55_13520,MB02_RS00150 and KC8_05330, in C. tardaugens NBRC 16725 (CT), A. estronivorus MHB5 (AE) and Sphingomonas sp. KC8 (KC8), respectively. DNA-binding domains ( a , c ) and effector-binding domains ( b , d ). Identical and conserved amino acids are marked with "*" and “:”, respectively. Genes 2021,12, 1846 8 of 20 To study the role of EdcR in the metabolism of E2 in C. tardaugens, we constructed a ∆ edcR deletion mutant that was cultured it in minimal medium supplemented with E2 as the sole carbon and energy source. The ∆ edcR strain is able to grow in E2 faster than the wild-type strain (Figure 6a), suggesting that EdcR is not an activator of E2 degradation. When the ∆ edcR mutant was complemented with plasmid pSEVA23edcR expressing the gene edcR under the control of the P lexA constitutive promoter (Table 1), the recombinant strain grew slower than the ∆ edcR mutant (Figure 6b), suggesting a probable repressor activity of the EdcR protein. Genes 2021, 12, x FOR PEER REVIEW 9 of 21 Figure 5. Sequence alignment of the regulators encoded by EGO55_13520, MB02_RS00150 and KC8_05330, in C. tardaugens NBRC 16725 (CT), A. estronivorus MHB5 (AE) and Sphingomonas sp. KC8 (KC8), respectively. DNA-binding domains (a,c) and effector-binding domains (b,d). Identical and conserved amino acids are marked with "*" and “:”, respectively. To study the role of EdcR in the metabolism of E2 in C. tardaugens, we constructed a ∆edcR deletion mutant that was cultured it in minimal medium supplemented with E2 as the sole carbon and energy source. The ∆edcR strain is able to grow in E2 faster than the wild-type strain (Figure 6a), suggesting that EdcR is not an activator of E2 degradation. When the ∆edcR mutant was complemented with plasmid pSEVA23edcR expressing the gene edcR under the control of the P lexA constitutive promoter (Table 1), the recombinant strain grew slower than the ∆edcR mutant (Figure 6b), suggesting a probable repressor activity of the EdcR protein. Figure 6. Growth curves (OD 600 ) of C. tardaugens strains growing in minimal medium supplemented with 2 mM E2. (a) C. tardaugens wt strain and C. tardaugens ΔedcR mutant. (b) C. tardaugens ΔedcR (pSEVA23PlexA) (ΔedcR-v) and C. tardaugens ΔedcR (pSEVA23edcR) (ΔedcR-c). The values correspond to the mean of three independent biological replicas (n = 3) and error bars correspond to SD. The influence of the absence of edcR on the expression of the genes of the edc cluster was determined using the ∆edcR mutant strain. Total RNA was extracted from the cells of wild-type and ∆edcR mutant strains growing in minimal medium with E2 or TES as the only carbon and energy sources, and RT-PCRs were performed to determine the relative expression of some representative genes of the edc cluster. EGO55_13555, EGO55_13570 and EGO55_13600 genes were chosen as representative markers of the expression of the OpA and OpB operons. The housekeeping recA gene (EGO55_01665) was used as internal control. As expected, the expression of the genes was detected in the presence of E2 in the wild-type and ∆edcR mutant strains (Figure 7). However, in the presence Figure 6. Growth curves (OD 600 ) of C. tardaugens strains growing in minimal medium supplemented with 2 mM E2. ( a )C. tardaugens wt strain and C. tardaugens ∆ edcR mutant. ( b )C. tardaugens ∆ edcR (pSEVA23PlexA) ( ∆ edcR-v) and C. tardaugens ∆ edcR (pSEVA23edcR) ( ∆ edcR-c). The values correspond to the mean of three independent biological replicas (n= 3) and error bars correspond to SD. The influence of the absence of edcR on the expression of the genes of the edc cluster was determined using the ∆ edcR mutant strain. Total RNA was extracted from the cells of wild-type and ∆ edcR mutant strains growing in minimal medium with E2 or TES as the only carbon and energy sources, and RT-PCRs were performed to determine the relative expression of some representative genes of the edc cluster. EGO55_13555,EGO55_13570 and EGO55_13600 genes were chosen as representative markers of the expression of the OpA and OpB operons. The housekeeping recA gene (EGO55_01665) was used as internal control. As expected, the expression of the genes was detected in the presence of E2 in the wild-type and ∆ edcR mutant strains (Figure 7). However, in the presence of TES the expression was only detected in the ∆ edcR mutant strain (Figure 7). This result strongly reinforces the hypothesis that EdcR negatively regulates the expression of these edc genes and that the effector molecule could be E2 or some intermediate of the degradation pathway. Genes 2021, 12, x FOR PEER REVIEW 10 of 21 of TES the expression was only detected in the ∆edcR mutant strain (Figure 7). This result strongly reinforces the hypothesis that EdcR negatively regulates the expression of these edc genes and that the effector molecule could be E2 or some intermediate of the degradation pathway. Figure 7. Analysis by RT-PCR of the expression of some genes of the edc cluster in C. tardaugens wild-type and ∆edcR mutant growing in E2 and testosterone. Cis the control without RNA, gDNA is the control with genomic DNA and M is the 100 bp molecular weight marker. 3.3. Analysis of the EdcR Effectors As a tool to identify the effector of EdcR, we constructed transcriptional fusions using the gfp as a reporter gene in replicative plasmids. The P a and P b promoter regions were cloned in the plasmid pSEVA237 (Table 1). The cloned promoter regions comprise approximately 300 bp upstream from the ribosome binding site (RBS) of the EGO55_13565 and EGO55_13570 genes, respectively. The resulting plasmids, pSEVA237Pa (Figure S2) and pSEVA237Pb (Figure S3) were used to transform C. tardaugens wild type and ∆edcR mutant strains taking advantage that pSEVA237 is replicative in this strain. The resulting strains C. tardaugens (pSEVA237Pa), C. tardaugens (pSEVA237Pb), C. tardaugens ∆edcR (pSEVA237Pa) and C. tardaugens ∆edcR (pSEVA237Pb) were cultivated in NB rich medium supplemented with 2 mM E1 or with CDX as control, and the fluorescence was measured at 36 h. The experiment was carried out using E1 instead of E2, since E1 is the second metabolite of the E2 pathway (Figure 1), and we observed that the strain is also able to grow in E1 as the sole carbon and energy source, suggesting that E1 should act as an effector of the edc cluster. The fluorescence intensity in the wild-type strain increased in the presence of E1 but remained constant in the mutant strain (Figure 8). This result correlates with the previous observation that the expression in the wild strain is inducible while, as expected, the expression of gfp gene from the P b and P a promoters in the ∆edcR mutant is constitutive (Figure 8). This result confirmed that EdcR acts as a transcriptional repressor of OpA and OpB operons in C. tardaugens and that E1 or another intermediate metabolite of the pathway is capable of inducing the expression of the edc cluster. Figure 8. Fluorescence intensity of the transcriptional fusions of the P a and P b promoter regions measured in cultures of C. tardaugens (a) and C. tardaugens ∆edcR mutant (b) transformed with the plasmids pSEVA237Pa or pSEVA237Pb, grown in NB carbon and energy source, in the presence of CDX (control) (light colors) or E1 (inducer) (dark colors). The values correspond to the mean of three independent biological replicas (n = 3) and error bars correspond to SD. Figure 7. Analysis by RT-PCR of the expression of some genes of the edc cluster in C. tardaugens wild-type and ∆ edcR mutant growing in E2 and testosterone. Cis the control without RNA, gDNA is the control with genomic DNA and M is the 100 bp molecular weight marker. 3.3. Analysis of the EdcR Effectors As a tool to identify the effector of EdcR, we constructed transcriptional fusions using the gfp as a reporter gene in replicative plasmids. The P a and P b promoter regions were cloned in the plasmid pSEVA237 (Table 1). The cloned promoter regions comprise Genes 2021,12, 1846 9 of 20 approximately 300 bp upstream from the ribosome binding site (RBS) of the EGO55_13565 and EGO55_13570 genes, respectively. The resulting plasmids, pSEVA237Pa (Figure S2) and pSEVA237Pb (Figure S3) were used to transform C. tardaugens wild type and ∆ edcR mutant strains taking advantage that pSEVA237 is replicative in this strain. The resulting strains C. tardaugens (pSEVA237Pa), C. tardaugens (pSEVA237Pb), C. tardaugens ∆ edcR (pSEVA237Pa) and C. tardaugens ∆ edcR (pSEVA237Pb) were cultivated in NB rich medium supplemented with 2 mM E1 or with CDX as control, and the fluorescence was measured at 36 h. The experiment was carried out using E1 instead of E2, since E1 is the second metabolite of the E2 pathway (Figure 1), and we observed that the strain is also able to grow in E1 as the sole carbon and energy source, suggesting that E1 should act as an effector of the edc cluster. The fluorescence intensity in the wild-type strain increased in the presence of E1 but remained constant in the mutant strain (Figure 8). This result correlates with the previous observation that the expression in the wild strain is inducible while, as expected, the expression of gfp gene from the P b and P a promoters in the ∆ edcR mutant is constitutive (Figure 8). This result confirmed that EdcR acts as a transcriptional repressor of OpA and OpB operons in C. tardaugens and that E1 or another intermediate metabolite of the pathway is capable of inducing the expression of the edc cluster. Genes 2021, 12, x FOR PEER REVIEW 10 of 21 of TES the expression was only detected in the ∆edcR mutant strain (Figure 7). This result strongly reinforces the hypothesis that EdcR negatively regulates the expression of these edc genes and that the effector molecule could be E2 or some intermediate of the degradation pathway. Figure 7. Analysis by RT-PCR of the expression of some genes of the edc cluster in C. tardaugens wild-type and ∆edcR mutant growing in E2 and testosterone. Cis the control without RNA, gDNA is the control with genomic DNA and M is the 100 bp molecular weight marker. 3.3. Analysis of the EdcR Effectors As a tool to identify the effector of EdcR, we constructed transcriptional fusions using the gfp as a reporter gene in replicative plasmids. The P a and P b promoter regions were cloned in the plasmid pSEVA237 (Table 1). The cloned promoter regions comprise approximately 300 bp upstream from the ribosome binding site (RBS) of the EGO55_13565 and EGO55_13570 genes, respectively. The resulting plasmids, pSEVA237Pa (Figure S2) and pSEVA237Pb (Figure S3) were used to transform C. tardaugens wild type and ∆edcR mutant strains taking advantage that pSEVA237 is replicative in this strain. The resulting strains C. tardaugens (pSEVA237Pa), C. tardaugens (pSEVA237Pb), C. tardaugens ∆edcR (pSEVA237Pa) and C. tardaugens ∆edcR (pSEVA237Pb) were cultivated in NB rich medium supplemented with 2 mM E1 or with CDX as control, and the fluorescence was measured at 36 h. The experiment was carried out using E1 instead of E2, since E1 is the second metabolite of the E2 pathway (Figure 1), and we observed that the strain is also able to grow in E1 as the sole carbon and energy source, suggesting that E1 should act as an effector of the edc cluster. The fluorescence intensity in the wild-type strain increased in the presence of E1 but remained constant in the mutant strain (Figure 8). This result correlates with the previous observation that the expression in the wild strain is inducible while, as expected, the expression of gfp gene from the P b and P a promoters in the ∆edcR mutant is constitutive (Figure 8). This result confirmed that EdcR acts as a transcriptional repressor of OpA and OpB operons in C. tardaugens and that E1 or another intermediate metabolite of the pathway is capable of inducing the expression of the edc cluster. Figure 8. Fluorescence intensity of the transcriptional fusions of the P a and P b promoter regions measured in cultures of C. tardaugens (a) and C. tardaugens ∆edcR mutant (b) transformed with the plasmids pSEVA237Pa or pSEVA237Pb, grown in NB carbon and energy source, in the presence of CDX (control) (light colors) or E1 (inducer) (dark colors). The values correspond to the mean of three independent biological replicas (n = 3) and error bars correspond to SD. Figure 8. Fluorescence intensity of the transcriptional fusions of the P a and P b promoter regions measured in cultures of C. tardaugens ( a ) and C. tardaugens ∆ edcR mutant ( b ) transformed with the plasmids pSEVA237Pa or pSEVA237Pb, grown in NB carbon and energy source, in the presence of CDX (control) (light colors) or E1 (inducer) (dark colors). The values correspond to the mean of three independent biological replicas (n= 3) and error bars correspond to SD. To determine if E1 is the true inducer, and to exclude the possible implication of other intermediate metabolites produced further on the degradation pathway, we used the C. tardaugens ∆ edcA strain, lacking the E1-hydroxylating activity that cannot transform E1 in other metabolites (Figure 1) [ 23 ]. It is worth mentioning here that the induction effect of E2 cannot be tested directly in C. tardaugens and although E2 is added as an inducer, we are testing the effect of E1, as E2 is transformed by the strain. C. tardaugens ∆ edcA was cultured in minimal medium supplemented with TES as a carbon and energy source in the presence of E1, and total RNA was extracted. The expression of EGO55_13555,EGO55_13570 and EGO55_13600 genes was analyzed by RT-PCR. Figure 9shows that the edc genes tested are induced in the presence of E1, suggesting that E1 acts as the true inducer of the edc cluster in C. tardaugens. Genes 2021, 12, x FOR PEER REVIEW 11 of 21 To determine if E1 is the true inducer, and to exclude the possible implication of other intermediate metabolites produced further on the degradation pathway, we used the C. tardaugens ∆edcA strain, lacking the E1-hydroxylating activity that cannot transform E1 in other metabolites (Figure 1) [23]. It is worth mentioning here that the induction effect of E2 cannot be tested directly in C. tardaugens and although E2 is added as an inducer, we are testing the effect of E1, as E2 is transformed by the strain. C. tardaugens ∆edcA was cultured in minimal medium supplemented with TES as a carbon and energy source in the presence of E1, and total RNA was extracted. The expression of EGO55_13555, EGO55_13570 and EGO55_13600 genes was analyzed by RT-PCR. Figure 9 shows that the edc genes tested are induced in the presence of E1, suggesting that E1 acts as the true inducer of the edc cluster in C. tardaugens. Figure 9. Analysis by RT-PCR of the expression of the genes of the edc cluster in C. tardaugens ∆edcA mutant growing in testosterone (TES) and testosterone in the presence of E2 (E2). Cis the control without RNA, gDNA is the control with genomic DNA and M is the 100 bp molecular weight marker. 3.4. Analysis of the Promoters of the edc Cluster in E. coli. Construction of an Estrogen Biosensor To monitor the expression from the P a and P b promoters in a heterologous host, we used the pSEVA237Pa and pSEVA237Pb plasmids carrying the gfp gene under the control of P a and P b promoters, respectively, able to replicate in E. coli. The recombinant strains of E. coli DH10B (pSEVA237PlexA) (control plasmid), E. coli DH10B (pSEVA237Pa) and E. coli DH10B (pSEVA237Pb) were grown on LB and the fluorescence intensity of the cells were analyzed after 5 h. The fluorescence signal was only detected in E. coli DH10B (pSEVA237Pb), suggesting that only the P b promoter is recognized by the E. coli RNApolymerase (Figure S4). To determine if EdcR can be a functional repressor of P b promoter in E. coli, the edcR gene was cloned under the control of the P lexA promoter in the plasmid pSEVA651 (Table 1), generating the plasmid pSEVA65edcR. This plasmid was transformed in E. coli DH10B (pSEVA237Pb), generating the strain E. coli DH10B (pSEVA237Pb, pSEVA65edcR). E. coli DH10B (pSEVA237Pb, pSEVA651) carrying the empty vectors was used as a control. When the strains were grown in LB and the fluorescence intensity was measured after 5 h, we observed that the production of EdcR decreases the fluorescence intensity, suggesting that EdcR acts as a transcriptional repressor on the P b promoter in E. coli (Figure 10). To confirm that E1 is the true inducer of the edc cluster, we tested its effect in E. coli DH10B (pSEVA237Pb, pSEVA65edcR). A partial recovery of the fluorescence signal was observed in the presence of E1, indicating that E1 is also able to induce the expression of P b promoter in E. coli (Figure 10). Interestingly, E2 had the same effect as E1, suggesting that E2 can be also recognized by EdcR as an inducer (Figure 10). A 2.9and 2.6-fold increase in signal was observed when E1 and E2 inducers were used, respectively, compared to solvent control. As mentioned above, this induction effect of E2 could not be tested in C. tardaugens since a mutant unable to transform E2 into E1 is not available [22]. Figure 9. Analysis by RT-PCR of the expression of the genes of the edc cluster in C. tardaugens ∆ edcA mutant growing in testosterone (TES) and testosterone in the presence of E2 (E2). Cis the control without RNA, gDNA is the control with genomic DNA and M is the 100 bp molecular weight marker. Genes 2021,12, 1846 16 of 20 26. Bertani, G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 1951 ,62, 293–300. [CrossRef] 27. Sambrook, J.; Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: New York, NY, USA, 2001. 28. Schafer, A.; Tauch, A.; Jager, W.; Kalinowski, J.; Thierbach, G.; Puhler, A. 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