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Sequence analysis inPseudomonas putida DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by thettgDEF genes, which are adjacent to thetod genes. ThettgDEF 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 inP. putida , 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 Tn5 insertion in thettgABC gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out thettgD 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 singlettgD mutant, DOT-T1E-1, and a double mutant with knock outs in thettgD andttgA 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 singlettgD mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type andttgD 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 inP. putida DOT-T1E.

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