G. Mosqueda et Jl. Ramos, A set of genes encoding a second toluene efflux system in Pseudomonas putida DOT-T1E is linked to the tod genes for toluene metabolism, J BACT, 182(4), 2000, pp. 937-943
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.