Complexity in efflux pump control: cross-regulation by the paralogues TtgV and TtgT

Pseudomonas putida DOT-T1E, known for its high tolerance to solvents, possesses three Resistance– Nodulation–Cell Division-type (RND) efflux pumps, namely TtgABC, TtgDEF and TtgGHI, which are involved in the active extrusion of solvents. Expression of the ttgABC and ttgGHI operons was previously shown to be regulated by the adjacently encoded repressors, TtgR and TtgV, respectively. Upstream of the third RND operon, ttgDEF, is located a putative regulator gene, ttgT. In this study, TtgT is shown to bind to the promoter region of the ttgDEF operon, and to be released from DNA in the presence of organic solvents. In vitro studies revealed that TtgV and TtgT bind the same operator sites in both the ttgDEF and the ttgGHI promoters. However, the affinity of TtgV for the ttgDEF operator was higher than that of TtgT, which, together with the fact that the ttgV promoter seems to be almost twice stronger than the ttgT promoter, explains why TtgV takes over in the regulation of the two efflux pump operons. The functional replacement of the cognate, chromosomally encoded TtgT by the plasmid-encoded paralogue TtgV illustrates a new mode of efflux pump regulation of which the physiological relevance is discussed.This work was supported by Grant QLK3-CT2002-01923 from the European Commission and Grant BIO2006-05668 from the Spanish Comisión Interministerial de Ciencia y Tecnología to J.L.R. and Grant RGY0021/2002 from the Human Frontier Science Programme to M.-T.G. W.T. was the recipient of a fellowship from the regional government of Andalusia, Spain (Junta de Andalucía).Peer reviewe