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Bacterial Sensor Kinase Tods Interacts with Agonistic and Antagonistic Signals

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Bacterial Sensor Kinase Tods Interacts with Agonistic and Antagonistic Signals Bacterial sensor kinase TodS interacts with agonistic and antagonistic signals Andreas Busch*, Jesu´ s Lacal*, Ariadna Martos†, Juan L. Ramos*‡, and Tino Krell* *Department of Environmental Protection, Estacio´n Experimental del Zaidı´n,Consejo Superior de Investigaciones Cientı´ficas,C/Profesor Albareda, 1, 18008 Granada, Spain; and †Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´ficas,C/Ramiro de Maetzu, 9, 28040 Madrid, Spain Edited by Thomas J. Silhavy, Princeton University, Princeton, NJ, and approved June 26, 2007 (received for review February 23, 2007) The TodS/TodT two-component system controls expression of the an increasing number of TCSs have been able to recognize and toluene dioxygenase (TOD) pathway for the metabolism of toluene respond to structurally different agonists. This finding is exempli- in Pseudomonas putida DOT-T1E. TodS is a sensor kinase that fied by PhoQ, which was initially reported to recognize bivalent ultimately controls tod gene expression through its cognate re- cations (9), although recent studies showed that PhoQ also binds sponse regulator, TodT. We used isothermal titration calorimetry cationic antimicrobial peptides (10, 11) by the same binding site for to study the binding of different compounds to TodS and related both types of agonists (12). Signal-recognition HPKs are generally these findings to their capacity to induce gene expression in vivo. assumed to trigger a regulatory response. However, in the light of Agonistic compounds bound to TodS and induced gene expression data showing that some HPKs recognize a wide range of chemical in vivo. Toluene was a powerful agonist, but ortho-substitutions of signals, it is of interest to establish whether the binding of a ligand toluene reduced or abolished in vivo responses, although TodS at the sensor domain of an HPK is enough to trigger this kind of recognized o-xylene with high affinity. These compounds were response. called antagonists. We show that agonists and antagonists com- We have attempted to shed light on these issues by investi- pete for binding to TodS both in vitro and in vivo. The failure of gating the TodS HPK in Pseudomonas putida, which forms a TCS antagonists to induce gene expression in vivo correlated with their with the TodT RR (13, 14). This TCS controls the expression of inability to stimulate TodS autophosphorylation in vitro. We pro- the toluene dioxygenase (TOD) pathway (15) responsible for the pose intramolecular TodS signal transmission, not molecular rec- metabolism of toluene into Krebs cycle intermediates (16). The ognition of compounds by TodS, to be the phenomenon that catabolic genes of the TOD pathway form an operon that is determines whether a given compound will lead to activation of transcribed from the PtodX promotor (13–16). expression of the tod genes. Molecular modeling identified resi- The architecture of the 108-kDa HPK TodS is atypical and dues F46, I74, F79, and I114 as being potentially involved in the complex. TodS has two supradomains, each containing a periodic binding of effector molecules. Alanine substitution mutants of circadian-Ah receptor single-minded protein (PAS) sensor domain these residues reduced affinities (2- to 345-fold) for both agonistic and a histidine kinase domain (Fig. 1), which are separated by an and antagonistic compounds. Our data indicate that determining RR receiver domain. TodS lacks transmembrane regions and is thus the inhibitory activity of antagonists is a potentially fruitful alter- likely to be located in the cytosol (8, 13). The N-terminal PAS native to design specific two-component system inhibitors for the domain of TodS binds toluene with high affinity (KD Ϸ 700 nM) development of new drugs to inhibit processes regulated by (14). This binding increases the basal autophosphorylation rate of two-component systems. TodS, leading to transphosphorylation of TodT and transcription stimulation from PtodX (14). TodS seems to belong to a subfamily of histidine kinases ͉ isothermal titration calorimetry ͉ Pseudomonas ͉ HPKs involved in the control of catabolic pathways for the degra- two-component systems ͉ aromatic hydrocarbons dation of solvents. For example, TmoS (82% identity with TodS) controls toluene degradation by the T4MO pathway in Pseudomo- he most widely distributed type of transcriptional control in nas mendocina (17), TutC (49% identity) regulates the anaerobic Tprokaryotic microorganisms exposed to environmental cues degradation of toluene in Thaurea sp. strain T1 (18), and StyS (41% is the two-component regulatory system (TCS) (1). In fact, genes identity) in Pseudomonas sp. strain Y2 is involved in styrene encoding TCSs are present in almost all bacteria and typically degradation (19). represent Յ1% of their genomes (1, 2). TCSs are also present, In the present study, we used isothermal titration calorimetry although to a lesser extent, in archaea and eukaryotes such as (ITC) to measure the thermodynamic parameters for the binding of fungi, slime molds, and plants (3, 4). These systems are often a wide range of different compounds to purified TodS. We then made up of a histidine protein kinase (HPK) and a response related these data to the capacity of these compounds to induce regulator (RR). The recognition of physical or chemical signals gene expression in vivo and to their ability to stimulate TodS at the input domain of HPKs typically initiates modulation of its autophosphorylation activity in vitro. Almost all of the mono- and autophosphorylation activity. The phosphate of the HPK is bicyclic aromatics we analyzed bound to TodS. However, only some transferred to the RR, triggering alterations in the functional of these molecules induced gene expression in vivo, and this ability properties of its output domain and eventually leading to the stimulation of transcription. The HPK and their RR together Author contributions: J.L.R. designed research; A.B., J.L., and A.M. performed research; A.B. comprise a large and diverse group. Their diversity is particularly and A.M. contributed new reagents/analytic tools; A.B., J.L., J.L.R., and T.K. analyzed data; pronounced in the input domain of HPKs and the output domain and J.L.R. and T.K. wrote the paper. of RR, which were shown to belong to many different protein The authors declare no conflict of interest. families (1). This variety ensures that HPKs recognize many This article is a PNAS Direct Submission. different signals and RRs are involved in the regulation of Abbreviations: HPK, histidine protein kinase; ITC, isothermal titration calorimetry; PAS, different cellular processes (5–7). periodic circadian-Ah receptor single-minded protein; RR, response regulator; TCS, two- Although many TCSs have been studied so far, the primary component system; TMB, trimethylbenzene; TOD, toluene dioxygenase. environmental signals recognized by HPKs remain unknown for ‡To whom correspondence should be addressed. E-mail: [email protected]. most TCSs (8). This lack of information is often a consequence of This article contains supporting information online at www.pnas.org/cgi/content/full/ difficulties in expressing and purifying sensor kinases, which are 0701547104/DC1. membrane-bound in most cases (8). Furthermore, in recent years, © 2007 by The National Academy of Sciences of the USA 13774–13779 ͉ PNAS ͉ August 21, 2007 ͉ vol. 104 ͉ no. 34 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701547104 Downloaded by guest on September 27, 2021 We then investigated the influence of single substitutions on the aromatic ring [supporting information (SI) Fig. 6]. Styrene was found to be recognized by TodS with the highest affinity (KD ϭ 580 Ϯ 70 nM) among benzene derivatives carrying an aliphatic substitution. Ethyl, propyl, and butyl substitutions were recognized with lower affinity in comparison to benzene by a factor of 4, 23, and 110, respectively (Table 1). Ethylbenzene was found to be a weak Fig. 1. Domain organization of TodS. The NTodS and CTodS recombinant inducer in vivo, and propyl- and butylbenzene did not induce proteins are indicated. Agonists and antagonists bind to the PAS-1 domain. expression from PtodX in vivo (Table 1). Nitro-, chloro-, and flu- PAS, PAS-type sensor domain; HK, histidine kinase domain; RRR, response orobenzene bound to TodS with affinities in the low micromolar regulator receiver domain. range and were found to be potent inducers of expression from PtodX (Table 1). Benzamide and benzoate were not bound by TodS, which is consistent with their failure to induce gene expression in vivo. was related to their capacity to increase TodS autophosphorylation Taking into consideration that toluene is an efficient inducer in in vitro . vivo, the next set of experiments was aimed at evaluating the impact Results of toluene substitutions on the binding parameters. Initial experi- ments were carried out with the three xylene isomers (methyl- Comparison of the in Vitro Ligand Affinities of TodS and the Capacity substituted toluene). Surprisingly, all three xylenes bound to TodS of the Compounds to Induce Gene Expression in Vivo. Purified TodS with similar affinities (Fig. 2B and Table 1), but only m- and was subjected to microcalorimetric titration with different com- p-xylene were powerful inducers in vivo. In contrast, o-xylene failed pounds to determine the effector profile in vitro. In parallel, the to induce gene expression (Table 1). potential of these compounds to stimulate gene expression in vivo To verify whether the data recorded for the three xylenes ␤ was determined by measuring the -gal activity with a PtodX::lacZ represented a general pattern in toluene derivatives, TodS was fusion. The in vitro binding parameters and ␤-gal measurements are titrated with the three chlorotoluenes. Like xylenes, all three listed in Table 1. chlorotoluenes were bound by TodS, but only m- and p- ITC experiments revealed that TodS bound benzene with an chlorotoluene showed in vivo activity, whereas o-chlorotoluene was affinity as high as that of toluene (Table 1 and Fig. 2A), but not inactive in vivo (Table 1).
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