We previously constructed a lux-based biosensor system to investigate the binding of TetR to its DNA binding site (tetO), and to measure transcriptional induction elicited by tetracycline (Tc) and its derivatives [8, 10]. For the work described here, we sought to create a more sensitive Tc-responsive system. We placed the luxCDABE gene cluster under the control of a TetR-repressible promoter on a low copy number pSC101-derived plasmid [11]. This plasmid, named pYR
tetO
, mediated production of a high level of luminescence in E. coli (Figure 2A, white bars), while a plasmid containing the lux genes with no promoter (pYR) produced no detectable luminescence (Figure 2A black bars). TetR was then cloned into the same plasmid under the control of the arabinose-inducible pBAD promoter to produce pYR
tetOR
(Figure 1B). Expression of TetR in pYR
tetOR
led to repression of luminescence (Figure 2A, gray bars) caused by the binding of TetR to the tetO site in the promoter of the lux genes. Repression occurred even in the absence of arabinose, indicating that the small amount of TetR expression from the pBAD promoter in the absence of arabinose was sufficient for repression of lux expression, even though TetR expression could not be detected by western blot under these conditions. Addition of arabinose only led to a small reduction in luminescence (Figure 2B).
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) [12]. Anhydrotetracycline (Atc), a stronger inducer of TetR [13], 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.
To test the ability of the pYR
tetOR
system for in vivo detection of enzymatic activity against Tc, we investigated the TetX enzyme. TetX is an FAD-dependent monooxygenase from Bacteroides fragilis that has been shown to hydroxylate Tc creating an unstable compound that undergoes rapid decomposition [9]. To measure the effect of TetX when expressed in pYR
tetOR
-containing cells, we introduced a separate plasmid into these cells that expressed this enzyme (pET
tetX
, Figure 1B). For comparison, we also tested pYR
tetOR
-containing cells co-transformed with a plasmid expressing the D311A mutant of TetX (pET
tetXD
), which is substituted at a highly conserved residue in the FAD-binding site and was expected to possess no enzymatic activity (Additional file 1: Figure S1). We verified that the pET
tetX
construct produced active TetX and that the D311A mutant was reduced in activity using an in vitro fluorescence based assay for TetX activity (Additional file1: Figure S2). As shown in Figure 3A, we measured the luminescence generated by pET
tetX
– and pET
tetXD
-containing cells at varying concentrations of Atc, and found that considerably less light was emitted by cells containing the plasmid expressing the WT version of TetX. For example, pET
tetX
-bearing cells required a concentration of ~100 ng/mL Atc to generate a level of luminescence similar to that emitted from pET
tetXD
-containing cells at an Atc concentration of only 2 ng/mL. We surmised that the reduction in luminescence in pET
tetX
-containing cells was the result of TetX-mediated catalysis of Atc into an unstable product and/or a product that could no longer bind TetR. Thus, the concentration of Atc available within the cells to bind TetR was decreased, which led to a greater degree of transcriptional repression of the lux genes by TetR (Figure 1B). We found that a similarly large reduction of luminescence was elicited by pET
tetX
when the assay was done under conditions of high TetR expression (0.02% arabinose) even though induction did not occur until a much higher concentration of Atc was added (Figure 3B). Assays performed with Dox and ClTc indicated that, as expected [9], TetX was also active against these Tc derivatives since reduced luminescence was observed in pET
tetX
-containing cells treated with these compounds (Figures 3C, D). It should be noted that the expression levels of WT TetX and TetX-D311A were similar (Figure 3E), indicating that the luminescence differences observed above were due to differences in the activity of these enzymes. Similar reductions in luminescence were observed when cells were treated with Tc (data not shown).
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).
In our data fitting, the enzymatic activity of TetX on Dox and ClTc was modeled by entering the known in vitro Km and kcat values of TetX for these compounds as fixed parameters [9]. The only free parameter in the fitting process was an arbitrary diffusion constant, K that accounted for the diffusion properties of Tc derivatives. Although parameters for the diffusion of Tc into E. coli have been experimentally determined [14, 15], the value of K for our fitting could not be determined a priori because it is not known how the diffusional properties of Tc derivatives would change upon modification by TetX or how quickly the modified Tc derivatives might degrade within the cell. In fitting the data from each experiment, the data generated for TetXD311A was used as a reference to predict how much luminescence would be generated at a given effective concentration of drug in the absence of enzyme. It can be seen in Table 1 that we were able to fit the Dox and ClTc data effectively using the known kinetic parameters of TetX (R2 values equaled 0.90 and 0.66 for Dox and ClTc, respectively). To fit the curves generated using Atc, for which Km and kcat values for TetX were not known, we allowed Km and kcat to also be free parameters. Notably, we were still able to obtain good fits to our data and the parameters returned were similar to those in the other fits. These data suggest that TetX acts on Atc with similar kinetic parameters as on Dox and ClTc. The ability to obtain fits to different experiments with consistent enzyme parameter values supports our conclusion that the behavior of this system is the result of the enzymatic activity of TetX against Tc derivatives. The use of the data generated from the pET
tetXD
-containing cells as the reference curve requires that the time taken after drug addition for the intracellular drug concentration to reach equilibrium in pET
tetX
-containing cells (t) is short enough to not affect luminescence accumulation. With the enzyme parameters from the above data fitting, we were also able to estimate t (see Methods for details). As shown in Table 1, t was less than 10 min in all experiments, which is much shorter than the time at which luminescence was measured (2-4 hours).
