Molecular characterization of the transcriptional regulator ttgv in pseudomonas putida dot-t1e, a solvent tolerant strain

Résumé

Solvent-tolerance is an innate character of some microorganisms such as Pseudomonas putida DOT-T1E. The resistance mechanism of this strain is mainly due to the presence in its genome of three efflux pumps, TtgABC, TtgDEF and TtgGHI that remove toxic compounds from the cells. Expression of the ttgDEF and ttgGHI operons is modulated in vivo by the TtgV repressor. TtgV is an IclR family member able to bind with high affinity (in the nanomolar range) the same intergenic region in both promoters thanks to its HTH motif. In this study, we have focussed on the physiological role of TtgV, the identification of the binding mode of this repressor to the DNA target sequences, and on the 3D structure of the regulator as a means to have a structure in hand to explore domain intercommunication. Our protein-DNA interaction studies revealed a high degree of flexibility of TtgV. We have revealed the importance of a long ¿-helix connecting the two domains to produce surface contacts to generate dimers, which further produce tetramers through "crosshand-interactions". Residue 86 is a key amino acid in TtgV rearrangement for DNA, whereas residues Arg 98 and Glu 102 are relevant in maintaining interdomain cross-talk. Data on TtgV provide solid grounds for the understanding of IclR family regulators and provide a structural framework to understand the mechanism of transcriptional control of a large number of tetrameric repressors.