Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
© Yu et al. 2010
Received: 2 June 2010
Accepted: 11 September 2010
Published: 11 September 2010
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© Yu et al. 2010
Received: 2 June 2010
Accepted: 11 September 2010
Published: 11 September 2010
Increasing our understanding of antibiotic resistance mechanisms is critical. To enable progress in this area, methods to rapidly identify and characterize antibiotic resistance conferring enzymes are required.
We have constructed a sensitive reporter system in Escherichia coli that can be used to detect and characterize the activity of enzymes that act upon the antibiotic, tetracycline and its derivatives. In this system, expression of the lux operon is regulated by the tetracycline repressor, TetR, which is expressed from the same plasmid under the control of an arabinose-inducible promoter. Addition of very low concentrations of tetracycline derivatives, well below growth inhibitory concentrations, resulted in luminescence production as a result of expression of the lux genes carried by the reporter plasmid. Introduction of another plasmid into this system expressing TetX, a tetracycline-inactivating enzyme, caused a marked loss in luminescence due to enzyme-mediated reduction in the intracellular Tc concentration. Data generated for the TetX enzyme using the reporter system could be effectively fit with the known K m and k cat values, demonstrating the usefulness of this system for quantitative analyses.
Since members of the TetR family of repressors regulate enzymes and pumps acting upon almost every known antibiotic and a wide range of other small molecules, reporter systems with the same design as presented here, but employing heterologous TetR-related proteins, could be developed to measure enzymatic activities against a wide range of antibiotics and other compounds. Thus, the assay described here has far-reaching applicability and could be adapted for high-throughput applications.
Over many decades, a wide variety of in vitro and in vivo screens have been used to identify small molecules with useful activities, such as antibiotics and enzyme inhibitors. However, there is still a need for simple and widely applicable assay systems for characterizing the activity of enzymes against specific small molecules that avoid the necessity of enzyme purification or high level expression. In the work described here, we have developed an in vivo luminescence-based reporter system that can be used to detect and characterize enzymatic activities against the antibiotic, Tetracycline (Tc). In the future, systems designed on the same principle could be used to investigate enzymes active against a variety of other small molecules.
Tc and its derivatives are highly effective broad specificity antibiotics that have been widely used for many decades . The ubiquitous utilization of tetracyclines has resulted in the emergence of numerous resistance mechanisms mediated by a variety of proteins including efflux pumps, drug modifying enzymes, and ribosome protection factors . The significant negative clinical impact of resistance to tetracyclines has led to intensive efforts to elucidate the mechanisms of this resistance and to develop new Tc derivatives that will overcome resistance mechanisms. To this end, much research has focused on enzymes capable of modifying tetracycline derivatives, either as a resistance mechanism or as a step in the tetracycline synthesis process . It is hoped that characterization of these enzymes will lead to approaches for combating resistance and creating more potent tetracycline derivatives. Although in vitro spectroscopic methods are available to assess some enzymatic modifications of tetracyclines, in vivo assays of these enzymes are quite complicated if the enzyme activity does not confer resistance to the growth inhibitory effect of the antibiotic . To aid in characterizing enzymes that modify tetracyclines and in identifying novel enzymes active against tetracyclines, a simple in vivo method to detect these activities would be very useful.
In a previous study, we designed a TetR-based biosensor that produced luminescence upon addition of tetracycline derivatives . The goals of the work described here were to improve the sensitivity of this system and to then exploit it to detect the activities of tetracycline modifying enzymes. To this end, a TetR regulated transcriptional promoter was cloned upstream of the lux operon in such a way that luminescence was elicited at very low concentrations of Tc. We then demonstrated that introduction of the Tc-modifying enzyme, TetX , into this system led to a significant reduction in luminescence due to the activity of the enzyme, which degrades the inducer of TetR. This reporter system could be modified to both identify ligands for TFRs of unknown function, and to detect enzymes active against these ligands. Since there are currently at least 50 known ligands for TFRs , the assay principle described here is applicable to the characterization of a considerable number enzymes active against small molecules.
To demonstrate the utility of our system for detecting Tc and its derivatives, luminescence production from pYR tetOR was measured in the presence of varying concentrations of these antibiotics. In these experiments the degree of induction is expressed as an induction ratio, the luminescence generated from pYR tetOR divided by that from pYR tetO (Figure 2A, white bars). Significant luminescence production was observed at Tc concentrations as low as 1 ng/mL, which is far below its minimum inhibitory concentration (MIC) in E. coli of 500 ng/mL (Figure 2C, white bars) . Anhydrotetracycline (Atc), a stronger inducer of TetR , also displayed a greater ability to induce TetR in this assay, relieving repression of the lux operon at a concentration of only 0.1 ng/mL. The addition of 0.02% arabinose to the system, which greatly increases the intracellular concentration of TetR (Figure 2B), led to a requirement for much higher concentrations of Tc and Atc, as well as the Tc analogs doxycycline (Dox) and chlorotetracycline (ClTc), to relieve lux repression (Figure 2D). For example, full induction with Atc under these conditions required a concentration of 200 ng/mL, indicating that minimizing the cellular concentration of TetR increases the sensitivity of the reporter system. Concentrations of Tc high enough to induce luminescence under conditions of increased TetR expression caused significant inhibition of cell growth (data not shown). Together, these data demonstrate that the pYR tetOR system can detect very low concentrations of Tc and its derivatives. The reporter system is sensitive to the concentration and chemical properties of inducer molecules, and to the intracellular concentration of TetR.
Our success in detecting the enzymatic activity of TetX in a cell based assay prompted us to determine whether the behavior observed in our assays could be accounted for by the known kinetic parameters of the TetX enzyme. To this end we formulated a series of equations to describe the TetX enzymatic activity within the cell based system in terms that were as simple as possible (see Methods for details). The objective of our analysis was to account for the difference between the dose-response curves generated in the presence of TetX as compared to TetXD311A, which we showed above is an inactive enzyme. Our equations were predicated on the assumption that once tetracycline is added, the media becomes an infinite drug reservoir. In the absence of TetX, drug molecules enter cells from the media driven by diffusion and rapidly reach an effective steady state concentration, which is referred to as [I eff ]. With TetX present inside the cell, the intracellular concentration of drug is simultaneously increased by the process of diffusion and decreased by the enzymatic activity of TetX. The intracellular drug concentration reaches equilibrium only when the rate of inward diffusion is matched by the rate of enzymatic modification. Thus, the final effective concentration of drug within these cells ([I eff ]) is a function not only of the extracellular concentration of drug ([I out ]), but also the rate of drug diffusion, the enzymatic activity of TetX, and the time taken after drug addition for the intracellular drug concentration to reach equilibrium (t).
Fitting results of cell based TetX activity experimentsa
K m (μM)
k cat (s-1)
In the work described here we have developed a sensitive luminescence-based in vivo assay to detect enzymatic activity against tetracyclines. Our assay system is based on the ability of these compounds to induce TetR regulated transcription and the diminution of this induction that occurs when a Tc modifying enzyme activity is present within the cell. An important aspect of this system is that it not only detects enzyme activity, but also allows quantitation of this activity. The only requirement is that the Tc derivative is modified in a way that lowers its affinity for TetR. Of course, our system will fail to detect an enzyme activity if the modification produced does not change the affinity of the Tc derivative for TetR. However, due to the sensitivity of our assay system and the use of titrations to detect activity, even a small change in affinity would be detected. In addition, there are a number of TetR mutants with varying reactivities towards different Tc derivatives [16, 17] that could be utilized in our assay system to maximize the number of Tc modifications that could be detected. Finally, there are other TetR-like repressors that are induced by Tc derivatives and at least one of these, TtgR of Pseudomonas putida , binds to Tc by a completely different mechanism compared to TetR. By using these different repressors, a wide range of modifications of tetracyclines would likely be detectable. Since there is great interest in the identification of enzymes that may modify tetracyclines to produce more potent antibiotics , our assay could be useful as a rapid screen to determine the level of activity of a given enzyme against a range of tetracyclines. Because of the wide range of ligands bound by TFRs , the assay system described here could be adapted to test for enzymatic activities against a huge variety of antibiotics and other small molecules. Supporting the general utility of our system, we have constructed reporters analogous to pYR tetOR for 22 diverse TFRs, and all of these TFRs are able to repress transcription of the lux operon when expressed in E. coli (data not shown). A reporter similar to the one described here was used to discover new ligands for the ActR repressor of S. coelicolor [20, 21]. Future studies will determine whether enzyme activities for a variety of small molecules will be detectable using these systems. It should be noted that the same principles used to design this TetR-based system could be used for any of the other families of transcriptional regulators that are induced by small molecules.
In general, our TetR-based system or other systems designed in a similar manner present many advantages for investigation of enzymes with activities against small molecules. First, we are able to detect enzyme activity using only nanogram quantities of compound. This feature could be critical in screening for activities against compounds that are available only in small quantities as in the case of compound libraries used for high-throughput screening. The ability to modulate the expression level of the repressor in our system by addition of varying concentrations of arabinose allows the intracellular repressor concentration to be adjusted to a level at which there is just enough present to repress transcription. Consequently, addition of only a small amount of inducer is required for derepression of the system. A second advantage of our system is that it provides the potential to investigate enzymatic activities without having to purify the enzymes or know their co-factor requirements. Our assay system would be equally capable of measuring the activity of difficult to purify membrane proteins, such as drug efflux pumps. TetR-based reporter systems have been shown to function in a wide range of cell types including mammalian cells [22–24], thus, enzymatic assays operating by the same principle as ours could be adapted to many cell types. A final advantage of our system is that it could be used to screen for enzymes with activity against a given compound of interest. For this purpose, a library of plasmids expressing candidate enzymes would be transformed into a strain containing a pYR-derivative that responded to the compound of interest. Individual colonies could then be screened in a 96-well format for reduced luminescence resulting from expression of an enzyme that modified the TFR-binding molecule. In this way, it will be possible to systematically identify novel enzymes with activities against many important small molecules.
Bacterial strains and plasmids used in this work are shown in Additional file1: Table S1. E. coli cells were grown at 37°C in Luria broth (LB) or LB agar medium containing the following antibiotics when necessary: kanamycin (50 μg/mL), ampicilin (100 μg/mL) and streptomycin (50 μg/mL). Protein expression for purification and E. coli drug susceptibility assays were carried out in E. coli BL21*(DE3). In vivo repression and induction assays were performed using E. coli Top10 (Invitrogen) because it is an arabinose metabolism deficient strain and produces more luminescence than BL21*(DE3). A T7 polymerase bearing E. coli Top10 (DE3) strain was constructed for the in vivo enzymatic activity assay using the Lambda DE3 Lysogenization Kit from Novagen.
Standard procedures were employed for all DNA manipulation and molecular cloning . The oligonucleotides and primers used in this study were synthesized from the ACGT Corporation (Toronto) and listed in Additional file1: Table S2. PCR reactions were carried out using Vent DNA polymerase (New England Biolabs). The QuikChange site-directed mutagenesis protocol (Stratagene) was employed to create point mutations of TetX.
The pCS26-Pac plasmid , which contains the luxCDABE operon on a low copy number pSC101-derived vector , was modified by inserting a double stranded oligonucleotide (oligo YLuxupdateF annealed to oligo YLuxupdateR) upstream of luxCDABE operon in between XhoI and BamHI sites. The araBAD promoter and the araC gene from the pBAD vector (Invitrogen), were amplified using primers SR204 and SR205 and then ligated into the EcoRI and KpnI restriction sites introduced as described above, creating the pYR plasmid (~13 kbp). A synthetic promoter consisting of the TetR operator and promoter tetO from the Tn10 tetA promoter region was prepared by annealing YZ201 and YZ202 oligonucleotides. This promoter was then introduced into pYR between KpnI and PmlI upstream of the luxCDABE cluster to give pYR tetO . The tetR gene was amplified from pET tetR  using primer YZ203 and YZ204 and cloned into pYR tetO between XhoI and EcoRI, downstream of the araBAD promoter, to give pYR tetOR . Expression vector pET tetX was prepared by introducing the PCR amplified tetX gene using primer pair YZ241-2 YZ242 into pET21 between EcoRI and XhoI sites.
For the repression and induction assays, isolated E. coli colonies were used to inoculate 2 mL cultures, which were grown overnight. 10 μL of overnight culture was added into 2 mL of LB media in the presence of varying concentrations of arabinose and drugs. These cultures were grown for 12 to 16 h before measurement of luminescence using a BMG Fluostar OPTIMA luminometer. In the enzyme assays, we inoculated 4 μL overnight cultures into 200 μL fresh pH 7 buffered LB media containing 0.04% glycerol, then grew for 2-4 h until early log phase and added varying concentrations of drugs. Luminescence and optical density were measured every 15 min using a TECAN Infinite M200 luminometer. Luminescence from similar early stationary phase cells (around 2-4 h after drug induction) was used to calculate the induction ratio. Multiple replicates (N > = 2) were performed and the 95% confidence intervals which are 1.96 times standard errors were displayed as error bars.
where a, b, c and L 0 , are arbitrary parameters giving the completed standard curves. Although these equations have no physical relevance to the functioning of the system, they accurately captured the relationship between L and [I out ] under these conditions (R2 > 0.9 in every fitting). Finally the L versus [I out ] curves for pET tetX -containing cells were fitted to the following equations using the information derived from the fits of the pET tetXD -containing cells.
The equations above also took into account the competition for drugs from TetR. Since TetR binds Tc derivatives much more tightly than TetX, TetX will only induce when TetR is saturated. The TetR saturating inducer concentration [I s ] was set to be the inducer concentration corresponding to the onset of luminescence induction. For fitting curves to data derived from pET tetX -containing cells in response to Dox and ClTc, only the K value was left as free parameters and published in vitro K m and k cat values were used.
where v d was determined from equation (3) and v t was determined from equation (1). With K, K m and k cat determined in previous steps, t became the only free parameter and can be returned by data fitting using equation (7) or (8).
To estimate in vivo enzyme concentrations, TetX-bearing cells were harvested at the time when drugs were added to induce luminescence. Approximately 1 μL cell pellet was lysed and loaded on a SDS-PAGE and subjected to a Western blot using anti-6xHis antibody. A series of purified 6xHis tagged TetR was loaded on the same gel as concentration standards (Figure 3E). TetX was estimated to be 15 μM.
Proteins were expressed as C-terminal hexahistidine fusions in E. coli strain BL21*(DE3) using plasmids derived from pET21d (Novagen). TetR was purified as previously described . For TetX and mutants, cells bearing pET tetX were grown at 30°C to an OD600 of 0.8, induced with 1 mM IPTG, and the grown overnight at 20°C. Cells were harvested and lysed in 6 M GuHCl. Protein was purified using nickel affinity chromatography by the denatured protein procedure (Qiagen). TetX was refolded by dialyzing into 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 2% glycerol. These proteins were further purified through a hi-load sephadex-75 gel filtration column (Pharmacia) using a Pharmacia LKB FPLC. All in vitro protein assays were performed in the dialysis buffer described above.
Fluorescence assays were performed using an Aviv ATF 105 spectrofluorometer in a 1 cm path length cuvette. Complex solutions with the indicated concentration of ingredients were excited at 340 nm and emission between 350 and 600 nm was recorded over time, averaging for 2 seconds at each wavelength.
TetR family of transcriptional regulators
minimum inhibitory concentration
Funding was provided by operating grants from the Canadian Institutes of Health Research awarded to A.R.D. (MOP-13609) and J.R.N. (MOP-97729). Z.Y. was supported by an Ontario Graduate Scholarship and L.C. was supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. S.E.R was supported by a Canadian Institutes of Health Research Training Grant in Protein Folding. The authors would like to thank William Navarre for allowing us to use his TECAN Infinite F200 plate reader.
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