Cloning and identification of a new repressor of 3,17β-Hydroxysteroid dehydrogenase of Comamonas testosteroni

3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17β-HSD. Previous reports showed that three tetR genes are located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first tetR gene encoded a potential repressor of 3,17β-HSD by sensing environmental signals. However, whether the other proposed tetR genes act as repressors of 3,17β-HSD are still unknown. In the present study, we cloned the second tetR gene and analyzed the regulatory mechanism of the protein on 3,17β-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second tetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17β-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17β-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological genetic stability, and the expression of 3,17β-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction. The second tetR gene acts as a negative regulator in 3,17β-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment.

The tetracycline repressor (TetR) family contains a kind of transcriptional repressors [10,11]. TetR repressors show a characteristic helix-turn-helix (HTH) structural motif for binding to DNA operators inhibiting specific gene expression [10,12,13]. The regulatory network involved by the TetR family members can be simple or complex [10]. For example, TetR inhibits membrane-associated protein (TetA) transcription by binding to the target operator upstream of the tetA gene, while the expression of the TetR family member is regulated by another regulator through a series of regulatory cascades [10]. Moreover, the TetR family member also can trigger a cell response to react to environmental signals [10].

Reagents
Gold nanoparticles (AuNPs) was kindly provided by Prof Zhenxin Wang (National Analytical Research Center of Electrochemistry and Spectroscopy, Changchun, China).
Hind III, EcoR I, Nde I, BamH I, testosterone, and IPTG were purchased from Sangon Biotech (Shanghai, China). DNA marker and protein marker were purchased from Thermo (USA).
To evaluate the growth characteristics of the recombinant C. testosteroni, the recombinants and wild type strain were cultured for 12 h in LB media containing kanamycin (100 µg/mL) and ampicillin (100 µg/mL), and the growth curves were generated based on the absorbance of cultures on OD 595 .

ELISA of 3,17β-HSD
The expression level of 3,17β-HSD in the strains was examined to evaluate genetic stability according to the ELISA protocol described by Xiong et al. [5]. The standard curve of 3,17β-HSD was generated by examining the absorbance of the purified 3,17β-HSD protein (kindly provided by Prof. Xiong) was diluted to 1.95, 3.9, 7.813, 15.625, 31.5, 62.5, and 250 ng/mL. The mutant and wild type strains were cultured in LB containing kanamycin (100 µg/mL) and ampicillin (100 µg/mL), with or without 1 mmol/L testosterone for 12 h. The strains were subculture five generations in the same conditions. The level of 3,17β-HSD was examined using ELISA based on the standard curve mentioned above. Three replicates were used for each sample.

Cloning and expression of tetR gene
Primers were designed and synthesized according to the second tetR gene located on 135580-136239 (complement) of C. testosteroni ATCC 11996 contig58 (GenBank: AHIL01000049.1). Forward primer was 5'-CAA CAT ATG CTG CAT CTC ATG CAA-3' (BamH I was underlined), and the reverse primer was 5'-ATC GGA TCC TCA ACG CTC TCC AAT GAA TAAG-3' (Nde I was underlined). The tetR gene (660 bp) was amplified with these primers and cloned into pET-15b with Nde I and Bam HI, followed by identification via double digestion with NdeI and BamHI, and sequencing. The resulting plasmid was designated as pET-15b-TetR.
To obtain the TetR protein, the recombinant plasmid pET-15b-TetR was transformed into E. coli BL21(DE3). The positive colony was cultured in LB to OD 600 of 0.6, followed by induction with 0.5 mmol/L IPTG at 37 °C for 4 h, respectively. Thereafter, cells were centrifuged at 4 °C and 8000×g for 15 min. Centrifuged cells were resuspended in 10 times lysis buffer, disrupted by sonication (20 kHz, 30 × 5 s), and centrifuged at 4 °C and 8000×g for 15 min. Then, the supernatants were collected and examined with SDS-PAGE or purified using HisPur™ Ni-NTA Resin (Thermoscientific, USA) according to protocol described previously [15].
Moreover, 125nM TetR was 1:8 mol concentration ratio mixed with testosterone (1 mM) for 40 min, followed by incubation with 125nM probe 2 for 20 min. centrifuged at 14 000 rpm for 10 min. Then, the mixture was added in 0.5 nM AuNPs binding buffer (10 mM Tris-HCl, 80 mM KCl) and analyzed for UV-visible spectra.

Statistical analysis
Statistical analysis was conducted using GraphPad software 5.0 (SanDiego, USA) with a one-way (ANOVA). P value < 0.05 is considered as significant difference. For each group of independent analysis, at least 3 independent experiments were evaluated. The results are expressed as mean ± standard deviation (SD) of three replicates.

Cloning and expression of C. testosteroni tetR gene
The tetR gene located on 135,580-136,239 (complement) of C. testosteroni ATCC 11,996 contig58 was amplified and subcloned into expression plasmid pET-15b, resulting a recombinant plasmid pET-15b-TetR. The recombinant plasmid pET-15b-TetR can be digested into two fragments, including a 660-bp tetR gene and a 5708-bp vector (Fig. 1a). The recombinant plasmid pET-15b-TetR was further confirmed by sequencing.
To obtain TetR protein, the recombinant plasmid pET-15b-TetR was transformed in to E. coli BL21(DE3), followed by induction with IPTG. The optimized expression condition is 0.5 mmol/L IPTG, at 37 ℃ for 4 h (Supplemental Fig. 1). Thereafter, the protein was purified using affinity chromatography (Fig. 1b). The results suggested that the TetR protein was cloned and expressed sucessfully in E. coli BL21(DE3).

Construction of recombinant C. testosteroni with tetR gene mutated
The C. testosteroni with the second tetR gene mutated was generated via homologous recombination by electrically transforming the recombinant plasmid pCR2.1-TetR into C. testosteroni. As shown in Fig. 2a, after homologous integration, an additional 'G' after start codon ATG was inserted in the tetR gene of the knock-out mutant, which caused the frameshift of the gene. The positive clone was identified by PCR (Fig. 2b) and further confirmed by sequencing (data not shown). Moreover, the strains in lanes 2, 4 and 5 can not continue to grow after multiple subcultures, which should be false positive. Only strain in lane 1, 3 can be subcultured for multiple times and still be alive as a gene knockout strain. Therefore, the strain in lane 1 was used in the following studies.
Subsequently, the growth characteristics of the recombinant C. testosteroni were evaluated (Fig. 2c). As shown in Fig. 2c, no significant difference was observed in the recombinant C. testosteroni compared with that of the wild-type C. testosteroni, suggesting the deletion of tetR gene has little effect on the normal growth of the bacteria.

TetR protein decreases the levels of 3,17β-HSD in C. testosteroni
To examine the effect of TetR protein on the expression of 3,17β-HSD protein, the levels of 3,17β-HSD expressed in the mutant C. testosteroni and the wild type strains were evaluated. As shown in Fig. 3a, the expression of 3,17β-HSD in wild-type and mutant strains without 1 mM testosterone induction was less different, but the expression of 3,17β-HSD protein in mutant and wild-type strains under the testosterone induction is higher than that of the groups without the induction, and the expression of 3,17β-HSD protein in mutant strains is slightly higher than that of the wild strains. Therefore, it is speculated that TetR can reduce the expression of 3,17β-HSD in C. testosteroni, but cannot completely inhibit the expression, suggesting the TetR is an repressor of 3,17β-HSD. Meanwhile, testosterone can induce 3,17β-HSD of both wild-type and mutant strains.
Moreover, the mutant C. testosteroni were cultured continuously for five generations, and the expression of 3,17β-HSD in each two generations was detected (Fig. 3b). The expression of 3,17β-HSD in the mutant was stable during the continuous culture of five generations, which was slightly higher than that in wild-type cells. Taken together, the mutant strains present good genetic stability, and have potential in theoretical research and practical application.

TetR protein interacts with DNA upstream of 3,17β-HSD gene
As reported, proteins of the TetR family have been found in 115 genera of proteobacteria, cyanobacteria, and archaea, composing a complex regulatory network for gene expression [10]. Some TetRs can bind to target operators to inhibit transcription, while others may involve a series of regulatory cascades, in which the expression of TetR family members is regulated by another regulator, or TetR family members trigger cell response to environmental damage [10].
To further evaluate the effect of TetR on 3,17β-HSD expression, electrophoretic mobility shift assay (EMSA) was conducted to examine the binding of the TetR to the 3,17β-HSD DNA. Three fragments were selected, synthesized, and used as double stranded probes, respectively. Two probes were conserved core consensus sequences located 1100bp upstream of the 3,17β-HSD gene, while the third probe was a non-conserved sequence located 1100-bp upstream of the gene, which was used as a negative control. Thereafter, probes (45 µmol/L) were incubated with the purified TetR  in different proportions, followed by electrophoretic analysis. As shown in Fig. 4, with the increase of protein concentration, gel block appeared in the electrophoresis band, while the mobility shift of the negative control protein phaC (polyhydroxyalkanoate synthetase) was similar in three gels, suggesting the phaC did not interact with the three probes. The mobility shift of the DNA bands of probe 1 and probe 2 decreased gradually with the increase of TetR protein concentration, indicating that TetR protein could bind to probe 1 (Fig. 4a) and probe 2 (Fig. 4b). Moreover, smeary bands were detected Fig. 4a, b, indicating that the protein may form polymers and bind to the probes with the increase of protein concentration. The negative DNA probe 3 does not bind to either phaC protein or TetR protein (Fig. 4c). Therefore, it can be concluded that the TetR protein can bind to two palindromic sequences upstream of the 3,17β-HSD gene.

Interaction between the TetR protein and DNA upstream of 3,17β-HSD gene was further confirmed by AuNPs-based assay
It is known that AuNPs is stable in water and has a clear surface plasmon peak at 520 nm. When AuNPs was exposed to buffer solution containing 80 mM KCl, a large number of nanoparticles gathered, and the absorbance at 520 nm decreased, while the absorption peak appeared at 750 nm [17]. Therefore, gold nanoparticles (AuNPs)-based assay was performed to further confirm the above results. Firstly, we determined a suitable TetR protein concentration for DNA binding (Fig. 5a). As shown in Fig. 5a, the higher the TetR protein concentration, the more stable the particles were. Thus, 500 nM TetR was selected to detect the formation of protein-DNA complex. In addition, when the concentration of TetR protein is 500 nM, the ability of TetR-DNA complex to protect AuNPs is also enhanced with the increase of the concentration of DNA probe 2 (Fig. 5b). To examine the TetR-DNA complex formation, the TetR protein (500 nM) were incubated with probe 1, 2, and probe 3 at 1:1 mol ratio, respectively, followed by incubating with AuNPs. As shown in Fig. 5c, The AuNPs are more stable in the TetR-probe 1 (a) and TetR-probe 2 (b) groups compared with that of the TetR-negative probe (c) and buffer (d) groups, suggesting that protein-DNA complexes were formed between TetR and probes. The degree of stabilization is probe 2 > probe 1 > negative control DNA probe. Moreover, the insets in the upper right corner of Fig. 5c are the color photos of the respective AuNPs solutions. It can be observed that both a and b groups are pink with little difference, indicating that protein-DNA complex has a good protective effect on AuNPs, while c is blue, suggesting an incomplete protection. Group d is dark blue due to denaturation of AuNPs in buffer solution. Therefore, combining the results of EMSA (Fig. 4) and AuNPs-based assay, it can be concluded that probe 2 has higher apparent affinity than probe 1. Probe 2 was used in the following studies.
To further confirm these results, TetR protein was 1:8 mol concentration mixed with testosterone, followed incubation with probe 2. Then, the mixture was added in AuNPs binding buffer, and analyzed for UV-visible spectra assay. As shown in Fig. 5d, TetR-DNA complex can efficiently bind to AuNPs in the group without testosterone (b, blue line), while the protective effect of TetR-DNA complex on AuNPs was released after testosterone was added (a, pink line), indicating that testosterone inhibits interaction between TetR and target DNA.
In order to prove that the potential electrosteric effect produced by protein-DNA complex is the reason for the stability of AuNPs, the Zeta potential of the surface charge of AuNPs under different sample treatments was measured (Fig. 5e). TetR has a weak positive charge, and AuNPs coated with TetR showed a much lower negative charge density than AuNPs coated with citrate ions. When the TetR-DNA complex is formed and coated on the AuNPs, the particles get more negative charges from the double-stranded DNA (dsDNA) in the TetR-DNA complex, which leads to the enhanced stability of the AuNPs nanoparticles.
Taken together, these results demonstrate that the TetR protein of C. testosteroni interacts with DNA upstream of the bacterial 3,17β-HSD gene, thus negatively regulating the expression of the 3,17β-HSD gene.

Discussion
Transcriptional regulation is an important way of biological regulation, most of which is carried out by the interaction between cis-acting elements and trans-acting factors. Notably, the regulation of gene expression in bacteria is mainly worked at transcription level. Previous reports showed that three tetR genes were located in the contig58 of C. testosteroni ATCC 11,996 (GenBank: AHIL01000049.1), 79,551-80,072, 181,113-181,673 (complement), and 135,580-136,239 (complement) [14]. In this study, we cloned the second tetR gene located on 135,580-136,239 (complement) of C. testosteroni ATCC 11,996 contig58. The results demonstrate that the second tetR gene was 660-bp, encoding a 26 kD protein, which could be expressed in E. coli induced by 0.5 mmol/L IPTG at 27 ℃ for 4 h from pET-15b-TetR (Fig. 1).
To evaluate the effect of second tetR gene, the gene was knocked-out via homologous recombination. Expectedly, the mutated strain has little difference on the growth compared to that of the wild-type C. testosteroni (Fig. 2c), and the expression of 3,17β-HSD in wild-type and mutant strains . TetR was mixed with 8 times of testosterone (a) or without testosterone (b) for 40 min, followed by incubation with probe 2 for 20 min. Then, the mixture was added in AuNPs (0.5 nM) binding buffer (10 mM Tris-HCl, 80 mM KCl), and analyzed for UV-visible spectra assay. e Zeta potential of different AuNPs samples in water and buffer solution (10 mM Tris-HCl, 80 mM KCl). The experiment were repeated three times without testosterone induction is similar (Fig. 3a), suggesting the deletion of second tetR gene has little effect on the normal growth of the bacteria. However, a slight increase of 3,17β-HSD protein was observed in the mutant group compared with that of the wild-type group under the testosterone induction (Fig. 3a, b), suggesting that the second TetR is a repressor of 3,17β-HSD, and the repressor activity is further regulated by testosterone.
To further confirm this hypothesis, the effect of the second TetR on 3,17β-HSD expression was evaluated using EMSA and AuNPs-based assay. As results showed (Figs. 4,  5), the TetR can specifically interact with two probes (probe 1 and probe 2) derived from the conserved consensus sequence located 1100-bp upstream of the 3,17β-HSD gene, but not with the non-palindromic sequence (probe 3). However, this interaction can be disrupted by testosterone (Fig. 5d). Moreover, due to the large size and concentrated negative charge of protein-DNA complex, protein-DNA complex can be coated on negatively charged AuNPs to provide protection against salt (such as KCl)-induced aggregation through electrostriction protection [17]. When the TetR-DNA complex is formed and coated on the AuNPs, the AuNPs particles get more negative charges from the double-stranded DNA (dsDNA) in the TetR-DNA complex than that of the TetR coated AuNPs, resulting in the enhanced stability of the AuNPs nanoparticles which was similar to that of the AuNPs coated with citric acid (Fig. 5e). These results indicate that the TetR protein encoded by the second tetR gene of C. testosteroni acts as another negative regulator, which interacts with consensus DNA upstream of the bacterial 3,17β-HSD gene, thus negatively regulating the expression of the 3,17β-HSD gene. After induction of testosterone, the inhibitory effect weakened and the level of 3,17β-HSD increased.
Notably, TetR protein family contains a kind of transcriptional repressors. We and other group [1,2,14] proved that two of three tetR genes in the contig58 of C. testosteroni ATCC 11,996 (GenBank: AHIL01000049.1) encode repressors regulating the expression of the 3,17β-HSD gene. Moreover, it was reported that LuxR protein also acted as a repressor in the expression of the 3,17β-HSD gene [18,19]. However, these repressors can only partially inhibit the expression of the 3,17β-HSD gene, indicating that the expression of the 3,17β-HSD gene may be synergistically regulated by several regulatory factors. The detailed regulatory mechanism of the 3,17β-HSD gene remains to be elucidated, and further confirmatory experiments are still in progress.

Conclusions
In conclusion, we confirmed that the second tetR gene in the contig58 of C. testosteroni regulates the expression of 3,17β-HSD gene via binding to the conserved core consensus sequences located 1100-bp upstream of the 3,17β-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out has good biological genetic stability, and the expression of 3,17β-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction, suggesting the mutant generated in this study might be used to treat environmental pollution caused by steroid hormones.The further study can help us to explore the application of the tetR gene mutant.
Author contribution HX and XG conceived and designed research. XW and XQ conducted experiments. CL contributed new reagents or analytical tools. YY and GY analyzed data. HX and XW wrote the manuscript. All authors read and approved the manuscript.
Funding None.
Data availability All datasets for this study are included in the manuscript files.

Conflict of interest
The authors declare no competing interests.

Consent to publish
The Author state herein that this manuscript has not been published elsewhere and that it has not been submitted simultaneously for publication elsewhere.