TtgV Bound to a Complex Operator Site Represses Transcription of the Promoter for the Multidrug and Solvent Extrusion TtgGHI Pump

The TtgGHI efflux pump of Pseudomonas putida extrudes a variety of antibiotics and solvents. We show that the ttgGHI operon is transcribed in vitro and in vivo from a single promoter and not from two overlapping promoters as previously proposed. The expression of this promoter is controlled by the TtgV repressor, whose operator expands through four helical turns that overlap the -10 region of the promoter. We also show that TtgV is released from its operator on binding of effectors such as aliphatic alcohols. Mutational analysis of the ttgGHI promoter revealed that substitutions at -13, -12, and -8 yielded promoters that were unable to drive transcription whereas certain mutations at -9, -11, and -6 to -3 increased expression in vivo. The cause of the increased expression was either a decrease in the affinity of the TtgV protein for its operator or an increase in the affinity of RNA polymerase for the mutant promoters.