A Set of Genes Encoding a Second Toluene Efflux System in <i>Pseudomonas putida</i> DOT-T1E Is Linked to the <i>tod</i> Genes for Toluene Metabolism

<jats:title>ABSTRACT</jats:title> <jats:p> Sequence analysis in <jats:italic>Pseudomonas putida</jats:italic> DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the <jats:italic>ttgDEF</jats:italic> genes, which are adjacent to the <jats:italic>tod</jats:italic> genes. The <jats:italic>ttgDEF</jats:italic> genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in <jats:italic>P. putida</jats:italic> , site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn <jats:italic>5</jats:italic> insertion in the <jats:italic>ttgABC</jats:italic> gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the <jats:italic>ttgD</jats:italic> gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single <jats:italic>ttgD</jats:italic> mutant, DOT-T1E-1, and a double mutant with knock outs in the <jats:italic>ttgD</jats:italic> and <jats:italic>ttgA</jats:italic> genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the single <jats:italic>ttgD</jats:italic> mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type and <jats:italic>ttgD</jats:italic> mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in <jats:italic>P. putida</jats:italic> DOT-T1E. </jats:p>