Copyright © by National Academy of Sciences. Final full-text version of the paper available at: http://www.pnas.org/cgi/content/abstract/104/34/13774
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.
This work was supported by Ministry of Science and Education
(Spain) Grant BIO-2006-05668, European Union Grant Sysmo GEN-2006-
27750-CS-5B, and Junta de Andalucía Grant CVI-344
Peer reviewed