New insights into the activation of o-xylene biodegradation in Pseudomonas stutzeri OX1 by pathway substrates

Abstract

The regulation of the tou operon of Pseudomonas stutzeri OX1, for degradation of toluene and o-xylene via phenolic intermediates, has been faithfully reconstructed in vitro with purified proteins. The set-up included the prokaryotic enhancer-binding protein TouR, the sigma54-dependent PToMO promoter and the sigma54-containing RNA polymerase. With this system we prove that direct binding of 2-methylphenol (o-cresol) to TouR is the only regulatory step for activation of PToMO in response to aromatic effectors, thereby ruling out the involvement of other factors or a need for protein processing. In addition, we found that while TouR failed entirely to activate PToMO in the absence of inducers, the protein had per se a very significant ATPase activity, which was only moderately increased by o-cresol addition. The results presented here support the view that TouR-like proteins are particularly suitable as evolutionary assets to endow recently evolved pathways for the degradation of environmental pollutants with an optimal degree of transcriptional regulation.

Fig. 1. In vivo properties of the TouR protein. (A) Functional domains of TouR (563 amino acids) and its A-domain-deleted derivative TouRΔA. The signal reception N-terminal module (called the A domain) is connected to the central C activating module, which bears an NTP binding motif. The A domain is deleted in the TouRΔA protein at amino acid L226, as indicated. The D module at the carboxyl end of the regulator includes a helix–turn–helix fold for DNA binding. To facilitate protein purification, a His₆ tail was engineered at the N-terminus of either TouR variant. (B) Transcriptional activation in vivo of the σ⁵⁴-dependent Pu promoter by TouRΔA and TouR. The reporter strain E. coli CC118Pu-lacZ (bearing a chromosomally inserted lacZ fusion to the Pu promoter of the TOL plasmid of P. putida mt-2) was transformed with either pHNΔAR (touRΔA⁺) or pTRR6N (touR⁺). Each of the transformants was grown at 37°C in LB medium with ampicillin (200 µg/ml) until cultures reached an OD₆₀₀ of 1.4. o-cresol (1 mM) was then added and the incubation continued for 2 h. Accumulation of β-galactosidase in each strain, measured in Miller’s units (Miller, 1972), is shown.

Fig. 2. Monitoring ATPase activity of TouR and TouRΔA. Mixtures of ATP (0.02 µCi/µl [γ-³²P]ATP, 3.0 mM ATP), TouRΔA or TouR (500 nM), the 208 bp EcoRI–HindIII fragment of pTE218 (UAS DNA) or a 146 bp fragment containing the multicloning site of pUC18 (MCS; 250 nM) were incubated for 30 min at 30°C and spotted onto TLC slides to separate Pi from non-hydrolysed ATP. Where indicated, samples were amended with 100 µM of o-cresol. The figures for ATPase activities are expressed as the percentage of labelled Pi released from the total [γ-³²P]ATP added to the reaction and corrected with the background value of the controls (lanes 1–4). A value of 50% corresponds to a specific activity of ∼210 nmol of hydrolysed ATP/min/nmol of dimeric TouR or TouRΔA.

Fig. 3. Dose-dependent stimulation of the ATPase activity of TouR by o-cresol, m-cresol and p-cresol. TouR (500 nM) was mixed with ATP (0.02 µCi/µl [γ-³²P]ATP, 3 mM ATP) and UAS DNA (250 nM) in the presence of increasing concentrations of each of the cresols. After incubation for 30 min at 30°C, samples were spotted onto TLC plates as in Figure 2 to monitor ATP hydrolysis. Activities are expressed as the percentage of the hydrolysis of ATP achieved by TouR with 100 µM o-cresol added.

Fig. 4. In vitro activation of the σ⁵⁴-dependent P_ToMO promoter by TouRΔA or TouR. Supercoiled plasmid pTE218 bearing the P_ToMO sequence was mixed with 0.5 U of core RNAP of E. coli, 150 nM purified σ⁵⁴ factor from P. putida, 2.5 mM ATP and 250 nM TouRΔA or TouR and subjected to a multiple-round transcription reaction as explained in Methods. Where indicated, the inducer o-cresol was added to a final concentration of 100 µM. Transcription of the P_ToMO promoter in the template employed generates an mRNA of 311 nucleotides, which becomes apparent in the autoradiograph of the gel shown.