Study of the TmoS/TmoT two-component system: towards the functional characterization of the family of TodS/TodT like systems.
Bottom Line: Tightest ligand binding was observed for toluene (K(D) = 150 nM), which corresponds to the highest affinity measured between an effector and a sensor kinase.Other TmoS ligands, termed antagonists, failed to increase TmoS autophosphorylation, which resulted in their incapacity to stimulate gene expression in vivo.Among these ligands, toluene was the most potent inducer of both promoter activities.
Affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda 1, Granada, Spain. .Show MeSH
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Mentions: The basal activities of promoters PtodX and PtmoX was of 2 ± 1 and 15 ± 4 Miller units respectively. Effectors that caused activities larger than two times the basal activities were considered as compounds that induce gene expression. From the 54 compounds analysed, 22 compounds induced gene expression in both systems. A plot of the activities of both promoters in response to this set of 22 compounds is shown in Fig. 5. The effector profile of PtodX was only marginally larger than that of PtmoX as only two compounds that were weak inducers of PtodX, namely catechol (13 ± 2 MU) and m‐bromotoluene (9 ± 2 MU), failed to induce PtmoX. The remaining 30 compounds, which are listed in the legend to Fig. 5, neither induced PtodX nor for PtmoX. Among these 30 compounds were o‐xylene, o‐chlorotoluene and 1,2,4‐trimethylbenzene, which were shown to bind tightly to purified TmoS (Table 1) but which failed to increase its phosphorylation state (Fig. 4). To elucidate whether compounds that bind but do not induce gene expression reduce the toluene‐mediated upregulation in gene expression, beta‐galactosidase measurements with mixtures of toluene with o‐xylene or o‐chlorotoluene were conducted. Cultures of P. mendocina KR1 harbouring pMIR38 were grown in Luria–Bertani (LB) to an OD600 of 0.2, at which point 0.25 mM o‐chlorotoluene, o‐xylene or the corresponding volume of buffer was added to three cultures. At an OD600 of 0.5 0.25 mM toluene was added to these cultures and β‐galactosidase activity was measured after another 2 h of growth. Similarly to the analogous experiments reported for the TodS/TodT system (Busch et al., 2007) the presence of o‐xylene and o‐chlorotoluene reduced the toluene mediated gene expression to 55 ± 7% and 32 ± 6%, respectively, of the value obtained for the culture containing toluene only.
Affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda 1, Granada, Spain. .