Cloning and expression of C. testosteroni TetR gene
The TetR gene located on 135580-136239 (complement) of C. testosteroni ATCC 11996 contig58 was amplified and subcloned into expression plasmid pET-15b, resulting a recombinant plasmid pET-15b-TetR. As shown in Fig. 1A, the recombinant plasmid pET-15b-TetR can be digested into two fragments, including a 660-bp TetR gene and a 5708-bp vector. 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, followed by induction with IPTG. The Fig. 1B and C showed that the TetR protein can be expressed in the recombinant plasmid-transformed E. coli. The optimized expression condition is 0.5 mmol/L IPTG, at 27 ℃ for 4 h. Thereafter, the protein was purified using affinity chromatography (Fig. 1D). These results suggested that the TetR protein was cloned and expressed sucessfully in E. coli.
Construction of recombinant C. testosteroni with TetR gene mutated
As shown in Fig. 2A, the recombinant C. testosteroni with the second TetR gene mutated was generated via homologous recombination. Briefly, a 301-bp fragment from 5’ terminal of TetR gene with a G insertion after start codon ATG were amplified and subcloned into pK18 plasmid (kindly provided by Prof. Xiong) (15), resulting a recombinant plasmid pK18-TetR. The plasmid was identified by PCR (Fig. 2B) and sequencing (data not shown). Then, the plasmid pK18-TetR was electrically transformed into C. testosteroni, followed by selection using kanamycin and ampicillin (100 µg/mL). The positive clone was identified by PCR (Fig. 2C) and further confirmed by sequencing (data not shown).
Subsequently, the growth characteristics of the recombinant C. testosteroni was evaluated (Fig. 2D). As shown in Fig. 2D, 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 testosterone induction is not much 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 it, suggesting the TetR is an inhibitor 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 have good genetic stability, and have potential application 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 (14). 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 (14).
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 1100-bp 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, the mobility shift of the negative control protein phaC was similar in three gels, suggesting the phaC did not interact with the three probes. However, 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 and 4B, 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 (1). 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. To examine the TetR-DNA complex formation, the TetR protein (500 nM) were incubated with probe 1, 2, and probe 3 at 1:1 ratio, respectively, followed by incubating with AuNPs. As shown in Fig. 5B, 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. 5 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. 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. 5C). 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 mixed with 8 times of 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.