A set of genes encoding a second toluene efflux system in Pseudomonas putida DOT-T1E is linked to the tod genes for toluene metabolism

Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toluene e fflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to the rod genes. The ttgDEF genes were expressed in response to t he presence of aromatic hydrocarbons such as toluene and styrene in the cul ture medium. To characterize the contribution of the TtgDEF system to tolue ne tolerance in P, putida, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1 E-18. This mutant carried a Tn5 insertion in the ttg4BC gene cluster, which encodes a toluene efflux pump that is synthesized constitutively, For site -directed mutagenesis, a cassette to knock out the ttgD gene and encoding r esistance to tellurite was constructed in vitro and transferred to the corr esponding host chromosome via the suicide plasmid pKNG101, Successful repla cement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization, A single ttgD mutant, DOT-T1E-1, and a double muta nt with knock outs in the ttgD and ttgA genes, DOT-T1E-82, were obtained an d characterized for toluene tolerance. This was assayed by the sudden addit ion of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growi ng on Luria-Bertani (LB) medium (noninduced) or on LB medium,with toluene s upplied via the gas phase (induced). Induced cells of the single ttgD mutan t were more sensitive to sudden toluene shock than were the wild-type cells ; however, noninduced wild-type and ttgD 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 upo n sudden toluene shock if they had been previously induced. These results a re discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in P. putida DOT-T1E.