Structural characterization of transcriptional regulation of solvent tolerance in gram negative bacteria

Bacteria antibiotic resistance to diverse clinically used compounds is becoming a major health risk. One strategy of multidrug resistance is the active extrusion of toxic compounds out of the bacterial cell walls by efflux pumps. These pumps are often regulated at gene transcriptional level. Pseudomonas putida, one of. the most resistant bacteria, is used as a model system to study the gene regulations involved in multidrug resistance. P. putida exhibits resistance to organic molecules, antibiotics and plant antimicrobials by three efflux pumps. One of them contains three membrane proteins TtgABC coded in one operon. The regulation of the efflux pumps genes ttgABC is controlled by a DNA binding repressor protein TtgR that is coded adjacent to the efflux pump operon and transcribed divergently. TtgR forms a homo dimer and consists of a ligand binding domain and a helix tum helix DNA binding domain. TtgR senses and interacts with ligands, leading to its release from DNA and the transcription of the efflux pump genes. The crystal structures of TtgR in complex with chloramphenicol and tetracycline antibiotics, phloretin, quercetin and naringenin plant antimicrobials and the organic solvent butylparaben have .,been determined to high-resolutions. These structures allow us to identify ligand binding pockets of TtgR and explain its diverse ligand binding properties. The structural information are confinned and complement~ by biochemical studies including isothermal titration calorimetry (lTC). Pseudomonas aeruginosa is a main pathogenic invader of the lungs of cystic fibrosis patients and exhibits multidrug resistance by activation of $e efflux pumps. Mex.Z, a homology of TtgR, is shown to be one of the most frequently mutated genes in bacteria isolated from cystic fibrosis patients, highlighting its importance in bacterial adaptation and survival. We have determined the crystal structure of MexZ, allowing us to explain the structural consequences of some of the clinically important mutations.