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. 2007 Aug 21;104(34):13774-9.
doi: 10.1073/pnas.0701547104. Epub 2007 Aug 10.

Bacterial sensor kinase TodS interacts with agonistic and antagonistic signals

Andreas Busch  1 , Jesús Lacal, Ariadna Martos, Juan L Ramos, Tino Krell
Affiliations

Bacterial sensor kinase TodS interacts with agonistic and antagonistic signals

Andreas Busch et al. Proc Natl Acad Sci U S A. .

Abstract

The TodS/TodT two-component system controls expression of the toluene dioxygenase (TOD) pathway for the metabolism of toluene in Pseudomonas putida DOT-T1E. TodS is a sensor kinase that ultimately controls tod gene expression through its cognate response regulator, TodT. We used isothermal titration calorimetry to study the binding of different compounds to TodS and related these findings to their capacity to induce gene expression in vivo. Agonistic compounds bound to TodS and induced gene expression in vivo. Toluene was a powerful agonist, but ortho-substitutions of toluene reduced or abolished in vivo responses, although TodS recognized o-xylene with high affinity. These compounds were called antagonists. We show that agonists and antagonists compete for binding to TodS both in vitro and in vivo. The failure of antagonists to induce gene expression in vivo correlated with their inability to stimulate TodS autophosphorylation in vitro. We propose intramolecular TodS signal transmission, not molecular recognition of compounds by TodS, to be the phenomenon that determines whether a given compound will lead to activation of expression of the tod genes. Molecular modeling identified residues F46, I74, F79, and I114 as being potentially involved in the binding of effector molecules. Alanine substitution mutants of these residues reduced affinities (2- to 345-fold) for both agonistic and antagonistic compounds. Our data indicate that determining the inhibitory activity of antagonists is a potentially fruitful alternative to design specific two-component system inhibitors for the development of new drugs to inhibit processes regulated by two-component systems.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Domain organization of TodS. The NTodS and CTodS recombinant proteins are indicated. Agonists and antagonists bind to the PAS-1 domain. PAS, PAS-type sensor domain; HK, histidine kinase domain; RRR, response regulator receiver domain.
Fig. 2.
Fig. 2.
Microcalorimetric titration of TodS with different hydrocarbons. (A) (Upper) Heat changes for the titration of 12 to 13.2 μM TodS with 4.8-μl aliquots of 750 μM cyclohexane and 1.6-μl aliquots of 750 μM benzene and 500 μM 1-naphthol. (Lower) Integrated and corrected peak areas for the titration with benzene and 1-naphthol. (B) Heat changes (Upper) and integrated peak areas (Lower) for the titration of 10–12 μM TodS with 1.6-μl aliquots of the three xylenes. For clarity, raw titration data have been shifted arbitrarily on the y axis. Derived thermodynamic data are given in Table 1.
Fig. 3.
Fig. 3.
Inhibition of toluene-mediated induction of PtodX by o-xylene, o-chlorotoluene, and 1,2,3-TMB. Eleven 25-ml cultures of P. putida DOT-T1E harboring pMIR66 (containing todST) and pMIR77 (containing a PtodX::lacZ fusion) were grown in LB to a turbidity of 0.2 at 660 nm. Then, six cultures were exposed to o-xylene, o-chlorotoluene, or 1,2,3-TMB (asterisk) at 0.3 mM (hatched bars) or 1.5 mM (dotted bars). When the cultures reached a turbidity of 0.5, buffer (control culture), o-xylene, o-chlorotoluene, or 1,2,3-TMB (all at a final concentration of 0.3 mM) was added to four cultures without addition, and 0.3 mM toluene was added to the seven remaining cultures. The β-gal activity was measured 2 h later. Data are the means and corresponding standard errors derived from at least three independent assays, each done in triplicate.
Fig. 4.
Fig. 4.
The 3D model of the N-terminal signal sensor domain of TodS. Secondary structure elements are indicated by S (strand) and H (helix). The amino acids in the proposed effector-binding site, which were replaced with alanine residues, are shown in ball-and-stick mode. This model contains amino acids 43–164 of TodS.
Fig. 5.
Fig. 5.
Modulation of TodS autophosphorylation activity in vitro. TodS autophosphorylation activity with [32P]ATP was measured in the absence of signal (control) and in the presence of 100 μM toluene, o-xylene, 1,2,3-TMB, or o-chlorotoluene. (A) SDS/PAGE of TodS in the absence and presence of different ligands. (B) Densitometric analysis of the data in A. Data are the means of three independent assays. Linear fit of the points at 2.5 and 10 min was used to calculate relative rates of autophosphorylation. Filled square, control; open triangle, o-chlorotoluene; open square, trimethylbenzene; open diamond, o-xylene; filled diamond, toluene.
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